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Detoxification of bisphenol A and nonylphenol by purified extracellular laccase from a fungus isolated from soil
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 98, No. 1, 64–66. 2004
Detoxification of Bisphenol A and Nonylphenol by Purified Extracellular Laccase from a Fungus Isolated from Soil TAKAO SAITO,1*KATSUYA KATO,1 YOSHIYUKI YOKOGAWA,1 MASAKAZU NISHIDA,1 AND NOBUYOSHI YAMASHITA2 Ceramics Research Institute, National Institute of Advanced Industrial Science and Technology, 2268-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan1 and The Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba 305-8569, Japan 2 Received 12 December 2003/Accepted 20 April 2004
Purified laccase from a fungus (family Chaetomiaceae) was used for the enzymatic oxidation of bisphenol A and nonylphenol, endocrine-disrupting chemicals. It rapidly oxidized both chemicals in the absence of mediators and within 24 h their estrogenic activities were completely removed. [Key words: endocrine-disrupting chemicals, bisphenol A, nonylphenol, laccase, oxidation, detoxification]
(PDB; Difco, Detroit, MI, USA) containing coffeic acid (1 mM) and copper sulfate (31 mM). Its laccase was purified to homogeneity by two steps of ammonium sulfate precipitation and HiPrep 16/10 Q FF (Amersham Bioscience, Piscataway, NJ, USA) anion exchange column chromatography, as described elsewhere (9). The laccase treatment reaction mixture (200 ml) consisted of BPA (5 mM) or NP (5 mM) and the purified laccase (50 U/ml) in 50 mM phosphate buffer, pH 7.0. It also contained 5% dimethyl sulfoxide (DMSO) to facilitate the dissolving of BPA and NP. The reaction was carried out at 40°C for 1, 3, 6, 12, and 24 h. At the end of each period, the residues and metabolites of BPA and NP were extracted with 200 ml of ethyl acetate and used in the subsequent analysis. The residues of BPA and NP were analyzed quantitatively by high-performance liquid chromatography (HPLC; Shimadzu, Kyoto) with a Wakosil-II5C18HG column (Wako Pure Chemical Industries, Osaka). Isocratic elution was done with 85% methanol in water (for BPA) or 100% methanol (for NP) at the flow rate of 0.5 ml/min. Detection was at 254 nm. As shown in Table 1, BPA was rapidly oxidized by the laccase, 93.7% being removed within 1 h, 99.2% within 3 h, and 100% by 6 h. Based on catalytic efficiency (kcat/Km) (9), NP is considered more resistant to oxidation by strain I-4 laccase than BPA. In fact, NP was oxidized more slowly
There is considerable concern that certain chemical compounds may affect the reproductive systems of wildlife and humans by mimicking or interfering with the action of endogenous gonadal steroid hormones (1). These compounds have been named endocrine-disrupting chemicals (EDCs). So far, about 50 compounds have been identified as possible EDCs. Bisphenol A (2,2-bis(4-hydroxyphenyl)propane; BPA) and nonylphenol (NP), major components in the production of various consumer products, including plastic packing materials and detergents, are known EDCs. They have been released on a large scale and form unbiodegradable accumulations in the surrounding environment. Many researchers have been studying various chemical and enzymatic methods to remove such EDCs from the environment. The ligninolytic enzymes lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (polyphenoloxidase; EC 1.10.3.2) completely biodegrade lignin polymers. Because of the complex structure of lignin, its biodegradation system is considered highly nonspecific. Ligninolytic enzymes therefore have attracted attention as possible degraders of environmental pollutants that differ structurally (2). The usefulness of these enzymes in the environmental cleanup of EDCs through oxidative degradation has been reported (3–8). Like the other ligninolytic enzymes, laccase has broad substrate specificity, as well as the advantage of not requiring an addition of harmful hydrogen peroxide to the oxidative reaction. We previously isolated a new fungus, strain I-4 of the family Chaetomiaceae, which produces large amounts of extracellular laccase (9). We showed that it has fairly broad substrate specificity and is relatively easy to produce and purify, indicative that it should have useful industrial and environmental applications. We here describe its environmental use to detoxify BPA and NP. Strain I-4 was cultured for 7 d in potato dextrose broth
TABLE 1. Time courses of BPA and NP percentages of remaining after laccase treatment with and without a mediator NP -1 mM HBT +1 mM HBT -1 mM HBT 0 100a 100 100 1 6.3 ±1.1 3.8 ± 6.3 29.8 ± 14.9 3 0.8 ±0.2 0.5 ± 1.1 9.8 ± 7.1 6 0 0 2.9 ± 2.6 12 0 0 0.1 ± 0.1 24 0 0 0 a Initial concentration (5 mM) at 0 h was defined as 100%. Time (h)
* Corresponding author. e-mail: [email protected] phone: +81-(0)52-736-7275 fax: +81-(0)52-736-7400 64
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than BPA, 70.2% being removed within 1 h, 90.2% within 3 h, 97.1% within 6 h, 99.9% within 12 h, and 100% by 24 h (Table 1). The laccase from lignin-degrading basidiomycetes required the addition of 1-hydroxybenzotriazole (HBT) to improve the oxidation of BPA and NP (4). As shown in Table 1, strain I-4 laccase completely oxidized both BPA and NP without a mediator, no addition of HBT (1 mM) being needed, evidence that it is highly suited to the enzymatic oxidation of both compounds. Estrogenic activities of the residues and metabolites of BPA and NP were estimated in MCF-7-derived cell line MVLN cells obtained from Dr. Pons of Institute de la Santé et de la Recherche Medicale (INSERM), Montpellier, France. MVLN cells are stably transfected MCF-7 cells with a receptor gene, allowing expression of firefly luciferase enzyme under control of the estrogen-regulatory element of the Xenopus vitellogenin A2 gene (10). Ethyl acetate extracts obtained from the reaction mixtures after 0, 1, and 24 h were evaporated, and their residues dissolved in DMSO. The DMSO solutions were added to the MVLN cell cultures to final concentrations of 10–7 to 10–5 M. The estrogenic-activity assay of the MVLN cells was done as described elsewhere (11). Luminescence was measured with a luciferase assay kit (Promega, Madison, WI, USA) and a multilabel counter (Wallac 1420 ARVOSX; PerkinElmer Life & Analytical Sciences, Boston, MA, USA). After 6 h, a small amount of estrogenic activity was present in both extracts, whereas after 24 h no estrogenic activity was detectable at concentrations of 10–7 to 10–5 M, evidence that BPA and NP were completely oxidized to compounds lacking estrogenicactivity after 24 h of treatment with strain I-4 laccase (Fig. 1). There have been several studies done on the metabolism
FIG. 1. Dose response curves of estrogenic activities (luminescence) of the residues and metabolites of BPA (A) and NP (B) in reaction mixtures after treatment with the purified laccase from strain I-4 for 0, 1, and 24 h. Symbols: closed circles, 0 h; open circles, 1 h; closed triangles, 24 h.
NOTES
65
of BPA oxidized by laccase (7, 12), horseradish peroxidase (HRP) (13), and MnP (14). Uchida et al. (12) reported that the laccase reaction from the basidiomycete Trametes villosa may include polymerization of BPA to form waterinsoluble, high molecular-weight (MW) compounds, such as oligomers, followed by the addition of phenol moieties or degradation of the oligomer to release water-soluble low MW compounds. They identified one high MW compound as a BPA dimmer, 5,5'-bis-[1-(4-hydroxy-phenyl)-1-methylethyl]-biphenyl-2,2'-diol and one low MW compound as 4-isopropenylphenol. The HRP-catalyzed oxidation product of BPA also has been identified as 4-isopropenylphenol (13). In the oxidation of BPA by the MnP from the white-rot basidiomycete Pleurotus ostreatus, the water-soluble low MW compounds 4-isopropenylphenol, phenol, 4-isopropylphenol, and hexestrol are thought to be formed (3). We measured the UV absorption spectra of ethyl acetate extracts prepared from 24-h laccase-oxidized BPA and NP. The extremely low absorbance at 279 nm (Fig. 2) indicates there was a very small amount of water-soluble phenolic compounds in the reaction mixtures. The 4-isopropenylphenol released in the oxidation reaction of BPA probably was further oxidized by the laccase. Laccase oxidation of BPA but not of NP, resulted in formation of water-insoluble products. After ethyl acetate extraction, the laccase-oxidized BPA mixture was centrifuged at 12,000 rpm for 15 min. The precipitate obtained was washed three times with water and evaporated yielding the solid form. FT-IR analysis showed that the IR-spectrum of the precipitate had peaks at 831, 1217, 1508, 1653, 2964, and 3393 (1/cm) similar to those of BPA (data not shown), indicative that this precipitate was polymerized BPA produced by laccase oxidation. Because it was chloroform-insoluble, oxidation by strain I-4 laccase is assumed to yield
FIG. 2. UV absorption spectra of the residues and metabolites of BPA (A) and NP (B) in reaction mixtures after treatment with the purified laccase from strain I-4 for 0 and 24 h. Lines: thick, 0 h; fine, 24 h.
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more highly polymerized BPA than that by the T. villosa laccase as demonstrated by Uchida et al. (12). We appreciate the help of Dr. Kuruthachalam Kannan (New York State University at Albany) with the estrogenic-activity assay of the MVLN cells and thank Ms. Hatsumi Achiwa for her technical assistance.
REFERENCES 1. Colborn, T., Dumanoski, D., and Myers, J. P.: Our stolen future. Dutton, New York (1996). 2. Dec, J. and Bollag, J. M.: Effect of various factors on dehalogenation of chlorinated phenols and anilines during oxidative coupling. Environ. Sci. Technol., 29, 657–663 (1994). 3. Hirano, T., Honda, Y., Watanabe, T., and Kuwahara, M.: Degradation of bisphenol A by the lignin-degrading enzyme, manganese peroxidase, produced by the white-rot basidiomycete, Pleurotus ostreatus. Biosci. Biotechnol. Biochem., 64, 1958–1962 (2000). 4. Tsutsumi, Y., Haneda, T., and Nishida, T.: Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes. Chemosphere, 42, 271–276 (2001). 5. Tanaka, T., Tonosaki, T., Nose, M., Tomidokoro, N., Kadomura, N., Fujii, T., and Taniguchi, M.: Treatment of model soils contaminated with phenolic endocrine-disrupting chemicals with laccase from Trametes sp. in a rotating reactor. J. Biosci. Bioeng., 92, 312–316 (2001). 6. Michizoe, J., Goto, M., and Furusaki, S.: Catalytic activity
7.
8.
9.
10.
11.
12.
13.
of laccase hosted in reversed micelles. J. Biosci. Bioeng., 92, 67–71 (2001). Fukuda, T., Uchida, H., Takashima, Y., Uwajima, T., Kawabata, T., and Suzuki, M.: Degradation of bisphenol A by purified laccase from Trametes villosa. Biochem. Biophys. Res. Commun., 284, 704–706 (2001). Okazaki, S., Michizoe, J., Goto, M., Furusaki, S., Wariishi, H., and Tanaka, H.: Oxidation of bisphenol A catalyzed by laccase hosted in reversed micelles in organic media. Enzyme Microb. Technol., 31, 227–232 (2002). Saito, T., Peng, H., Kato, K., Okazaki, M., Inagaki, H., Maeda, S., and Yokogawa, Y.: Purification and characterization of an extracellular laccase of a fungus (family Chaetomiaceae) isolated from soil. Enzyme Microb. Technol., 33, 520–526 (2003). Pons, M., Gagne, D., Nicolas, J. C., and Mehtali, M.: A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules. BioTechniques, 9, 450–459 (1990). Balaguer, P., Fabienne Francois, F., Comunale, F., Fenet, H., Boussoux, A. M., Pons, M., Nicolas, J. C., and Casellas, C.: Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ., 233, 47–56 (1999). Uchida, H., Fukuda, T., Miyamoto, H., Kawabata, T., Suzuki, M., and Uwajima, T.: Polymerization of bisphenol A by purified laccase from Trametes villosa. Biochem. Biophys. Res. Commun., 287, 355–358 (2001). Sakuyama, H., Endo, Y., Fujimoto, K., and Hatana, Y.: Oxidative degradation of alkylphenols by horseradish peroxidase. J. Biosci. Bioeng., 96, 227–231 (2003).
Detoxification of Bisphenol A and Nonylphenol by Purified Extracellular Laccase from a Fungus Isolated from Soil TAKAO SAITO,1*KATSUYA KATO,1 YOSHIYUKI YOKOGAWA,1 MASAKAZU NISHIDA,1 AND NOBUYOSHI YAMASHITA2 Ceramics Research Institute, National Institute of Advanced Industrial Science and Technology, 2268-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan1 and The Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba 305-8569, Japan 2 Received 12 December 2003/Accepted 20 April 2004
Purified laccase from a fungus (family Chaetomiaceae) was used for the enzymatic oxidation of bisphenol A and nonylphenol, endocrine-disrupting chemicals. It rapidly oxidized both chemicals in the absence of mediators and within 24 h their estrogenic activities were completely removed. [Key words: endocrine-disrupting chemicals, bisphenol A, nonylphenol, laccase, oxidation, detoxification]
(PDB; Difco, Detroit, MI, USA) containing coffeic acid (1 mM) and copper sulfate (31 mM). Its laccase was purified to homogeneity by two steps of ammonium sulfate precipitation and HiPrep 16/10 Q FF (Amersham Bioscience, Piscataway, NJ, USA) anion exchange column chromatography, as described elsewhere (9). The laccase treatment reaction mixture (200 ml) consisted of BPA (5 mM) or NP (5 mM) and the purified laccase (50 U/ml) in 50 mM phosphate buffer, pH 7.0. It also contained 5% dimethyl sulfoxide (DMSO) to facilitate the dissolving of BPA and NP. The reaction was carried out at 40°C for 1, 3, 6, 12, and 24 h. At the end of each period, the residues and metabolites of BPA and NP were extracted with 200 ml of ethyl acetate and used in the subsequent analysis. The residues of BPA and NP were analyzed quantitatively by high-performance liquid chromatography (HPLC; Shimadzu, Kyoto) with a Wakosil-II5C18HG column (Wako Pure Chemical Industries, Osaka). Isocratic elution was done with 85% methanol in water (for BPA) or 100% methanol (for NP) at the flow rate of 0.5 ml/min. Detection was at 254 nm. As shown in Table 1, BPA was rapidly oxidized by the laccase, 93.7% being removed within 1 h, 99.2% within 3 h, and 100% by 6 h. Based on catalytic efficiency (kcat/Km) (9), NP is considered more resistant to oxidation by strain I-4 laccase than BPA. In fact, NP was oxidized more slowly
There is considerable concern that certain chemical compounds may affect the reproductive systems of wildlife and humans by mimicking or interfering with the action of endogenous gonadal steroid hormones (1). These compounds have been named endocrine-disrupting chemicals (EDCs). So far, about 50 compounds have been identified as possible EDCs. Bisphenol A (2,2-bis(4-hydroxyphenyl)propane; BPA) and nonylphenol (NP), major components in the production of various consumer products, including plastic packing materials and detergents, are known EDCs. They have been released on a large scale and form unbiodegradable accumulations in the surrounding environment. Many researchers have been studying various chemical and enzymatic methods to remove such EDCs from the environment. The ligninolytic enzymes lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (polyphenoloxidase; EC 1.10.3.2) completely biodegrade lignin polymers. Because of the complex structure of lignin, its biodegradation system is considered highly nonspecific. Ligninolytic enzymes therefore have attracted attention as possible degraders of environmental pollutants that differ structurally (2). The usefulness of these enzymes in the environmental cleanup of EDCs through oxidative degradation has been reported (3–8). Like the other ligninolytic enzymes, laccase has broad substrate specificity, as well as the advantage of not requiring an addition of harmful hydrogen peroxide to the oxidative reaction. We previously isolated a new fungus, strain I-4 of the family Chaetomiaceae, which produces large amounts of extracellular laccase (9). We showed that it has fairly broad substrate specificity and is relatively easy to produce and purify, indicative that it should have useful industrial and environmental applications. We here describe its environmental use to detoxify BPA and NP. Strain I-4 was cultured for 7 d in potato dextrose broth
TABLE 1. Time courses of BPA and NP percentages of remaining after laccase treatment with and without a mediator NP -1 mM HBT +1 mM HBT -1 mM HBT 0 100a 100 100 1 6.3 ±1.1 3.8 ± 6.3 29.8 ± 14.9 3 0.8 ±0.2 0.5 ± 1.1 9.8 ± 7.1 6 0 0 2.9 ± 2.6 12 0 0 0.1 ± 0.1 24 0 0 0 a Initial concentration (5 mM) at 0 h was defined as 100%. Time (h)
* Corresponding author. e-mail: [email protected] phone: +81-(0)52-736-7275 fax: +81-(0)52-736-7400 64
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than BPA, 70.2% being removed within 1 h, 90.2% within 3 h, 97.1% within 6 h, 99.9% within 12 h, and 100% by 24 h (Table 1). The laccase from lignin-degrading basidiomycetes required the addition of 1-hydroxybenzotriazole (HBT) to improve the oxidation of BPA and NP (4). As shown in Table 1, strain I-4 laccase completely oxidized both BPA and NP without a mediator, no addition of HBT (1 mM) being needed, evidence that it is highly suited to the enzymatic oxidation of both compounds. Estrogenic activities of the residues and metabolites of BPA and NP were estimated in MCF-7-derived cell line MVLN cells obtained from Dr. Pons of Institute de la Santé et de la Recherche Medicale (INSERM), Montpellier, France. MVLN cells are stably transfected MCF-7 cells with a receptor gene, allowing expression of firefly luciferase enzyme under control of the estrogen-regulatory element of the Xenopus vitellogenin A2 gene (10). Ethyl acetate extracts obtained from the reaction mixtures after 0, 1, and 24 h were evaporated, and their residues dissolved in DMSO. The DMSO solutions were added to the MVLN cell cultures to final concentrations of 10–7 to 10–5 M. The estrogenic-activity assay of the MVLN cells was done as described elsewhere (11). Luminescence was measured with a luciferase assay kit (Promega, Madison, WI, USA) and a multilabel counter (Wallac 1420 ARVOSX; PerkinElmer Life & Analytical Sciences, Boston, MA, USA). After 6 h, a small amount of estrogenic activity was present in both extracts, whereas after 24 h no estrogenic activity was detectable at concentrations of 10–7 to 10–5 M, evidence that BPA and NP were completely oxidized to compounds lacking estrogenicactivity after 24 h of treatment with strain I-4 laccase (Fig. 1). There have been several studies done on the metabolism
FIG. 1. Dose response curves of estrogenic activities (luminescence) of the residues and metabolites of BPA (A) and NP (B) in reaction mixtures after treatment with the purified laccase from strain I-4 for 0, 1, and 24 h. Symbols: closed circles, 0 h; open circles, 1 h; closed triangles, 24 h.
NOTES
65
of BPA oxidized by laccase (7, 12), horseradish peroxidase (HRP) (13), and MnP (14). Uchida et al. (12) reported that the laccase reaction from the basidiomycete Trametes villosa may include polymerization of BPA to form waterinsoluble, high molecular-weight (MW) compounds, such as oligomers, followed by the addition of phenol moieties or degradation of the oligomer to release water-soluble low MW compounds. They identified one high MW compound as a BPA dimmer, 5,5'-bis-[1-(4-hydroxy-phenyl)-1-methylethyl]-biphenyl-2,2'-diol and one low MW compound as 4-isopropenylphenol. The HRP-catalyzed oxidation product of BPA also has been identified as 4-isopropenylphenol (13). In the oxidation of BPA by the MnP from the white-rot basidiomycete Pleurotus ostreatus, the water-soluble low MW compounds 4-isopropenylphenol, phenol, 4-isopropylphenol, and hexestrol are thought to be formed (3). We measured the UV absorption spectra of ethyl acetate extracts prepared from 24-h laccase-oxidized BPA and NP. The extremely low absorbance at 279 nm (Fig. 2) indicates there was a very small amount of water-soluble phenolic compounds in the reaction mixtures. The 4-isopropenylphenol released in the oxidation reaction of BPA probably was further oxidized by the laccase. Laccase oxidation of BPA but not of NP, resulted in formation of water-insoluble products. After ethyl acetate extraction, the laccase-oxidized BPA mixture was centrifuged at 12,000 rpm for 15 min. The precipitate obtained was washed three times with water and evaporated yielding the solid form. FT-IR analysis showed that the IR-spectrum of the precipitate had peaks at 831, 1217, 1508, 1653, 2964, and 3393 (1/cm) similar to those of BPA (data not shown), indicative that this precipitate was polymerized BPA produced by laccase oxidation. Because it was chloroform-insoluble, oxidation by strain I-4 laccase is assumed to yield
FIG. 2. UV absorption spectra of the residues and metabolites of BPA (A) and NP (B) in reaction mixtures after treatment with the purified laccase from strain I-4 for 0 and 24 h. Lines: thick, 0 h; fine, 24 h.
66
J. BIOSCI. BIOENG.,
SAITO ET AL.
more highly polymerized BPA than that by the T. villosa laccase as demonstrated by Uchida et al. (12). We appreciate the help of Dr. Kuruthachalam Kannan (New York State University at Albany) with the estrogenic-activity assay of the MVLN cells and thank Ms. Hatsumi Achiwa for her technical assistance.
REFERENCES 1. Colborn, T., Dumanoski, D., and Myers, J. P.: Our stolen future. Dutton, New York (1996). 2. Dec, J. and Bollag, J. M.: Effect of various factors on dehalogenation of chlorinated phenols and anilines during oxidative coupling. Environ. Sci. Technol., 29, 657–663 (1994). 3. Hirano, T., Honda, Y., Watanabe, T., and Kuwahara, M.: Degradation of bisphenol A by the lignin-degrading enzyme, manganese peroxidase, produced by the white-rot basidiomycete, Pleurotus ostreatus. Biosci. Biotechnol. Biochem., 64, 1958–1962 (2000). 4. Tsutsumi, Y., Haneda, T., and Nishida, T.: Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes. Chemosphere, 42, 271–276 (2001). 5. Tanaka, T., Tonosaki, T., Nose, M., Tomidokoro, N., Kadomura, N., Fujii, T., and Taniguchi, M.: Treatment of model soils contaminated with phenolic endocrine-disrupting chemicals with laccase from Trametes sp. in a rotating reactor. J. Biosci. Bioeng., 92, 312–316 (2001). 6. Michizoe, J., Goto, M., and Furusaki, S.: Catalytic activity
7.
8.
9.
10.
11.
12.
13.
of laccase hosted in reversed micelles. J. Biosci. Bioeng., 92, 67–71 (2001). Fukuda, T., Uchida, H., Takashima, Y., Uwajima, T., Kawabata, T., and Suzuki, M.: Degradation of bisphenol A by purified laccase from Trametes villosa. Biochem. Biophys. Res. Commun., 284, 704–706 (2001). Okazaki, S., Michizoe, J., Goto, M., Furusaki, S., Wariishi, H., and Tanaka, H.: Oxidation of bisphenol A catalyzed by laccase hosted in reversed micelles in organic media. Enzyme Microb. Technol., 31, 227–232 (2002). Saito, T., Peng, H., Kato, K., Okazaki, M., Inagaki, H., Maeda, S., and Yokogawa, Y.: Purification and characterization of an extracellular laccase of a fungus (family Chaetomiaceae) isolated from soil. Enzyme Microb. Technol., 33, 520–526 (2003). Pons, M., Gagne, D., Nicolas, J. C., and Mehtali, M.: A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules. BioTechniques, 9, 450–459 (1990). Balaguer, P., Fabienne Francois, F., Comunale, F., Fenet, H., Boussoux, A. M., Pons, M., Nicolas, J. C., and Casellas, C.: Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ., 233, 47–56 (1999). Uchida, H., Fukuda, T., Miyamoto, H., Kawabata, T., Suzuki, M., and Uwajima, T.: Polymerization of bisphenol A by purified laccase from Trametes villosa. Biochem. Biophys. Res. Commun., 287, 355–358 (2001). Sakuyama, H., Endo, Y., Fujimoto, K., and Hatana, Y.: Oxidative degradation of alkylphenols by horseradish peroxidase. J. Biosci. Bioeng., 96, 227–231 (2003).