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Enzymatic removal of phenols from aqueous solutions by Coprinus cinereus peroxidase and hydrogen peroxide
Journal of Biotechnology 73 (1999) 71 – 74 www.elsevier.com/locate/jbiotec
Short communication
Enzymatic removal of phenols from aqueous solutions by Coprinus cinereus peroxidase and hydrogen peroxide Carl Kauffmann a,*, B.R. Petersen b, Morten J. Bjerrum c a
The Otto Warburg Center for Biotechnology in Agriculture, Faculty of Agriculture, The Hebrew Uni6ersity of Jerusalem, 76100 Reho6ot, Israel b No6o Nordisk A/S, No6o Alle, 2880 Bags6aerd, Denmark c Chemistry Department, The Royal Veterinary and Agricultural Uni6ersity, Thor6aldsens6ey; 40, 1871 Frederiksberg C, Denmark Received 20 October 1998; accepted 29 March 1999
Abstract The fungal enzyme Coprinus cinereus peroxidase (CIP) can be used for the removal of toxic phenols from water. After treating aqueous solutions of phenols with CIP and H2O2 the phenols polymerized and precipitated. The decrease in phenol concentration was investigated for 10 different phenols. At neutral pH, the investigated phenols were in general removed with high efficiency. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Bioremediation; Coprinus cinereus peroxidase; Environmental biotechnology; Phenols
1. Introduction Various phenols are inherent in wastewaters from a number of industries. Nearly all phenols are toxic and some are known to be human carcinogens. Therefore removal of phenols from industrial aqueous effluents is of great practical significance (Klibanov et al., 1980; The Merck Index, 1996). Removal and/or detoxification of pollutants can be done by physical, chemical or biological * Corresponding author. Tel.: +972-8-948-1973; fax: + 972-8-946-8785. E-mail address: [email protected] (C. Kauffmann)
means, the latter being of special interest because of its potential low cost and low adverse effect on the environment. Because of their biocidic nature most phenols cannot be satisfactorily degraded during conventional biological wastewater treatment, and the presence of phenols in high concentrations may irreversibly shock and damage live microbial systems. Enzymatic wastewater treatment opens a new avenue for the treatment of effluent streams containing hazardous or xenobiotic organic pollutants. The use of enzymes allows the degradation to be carried out under mild biological conditions, and the rationale is to use free or immobilized
0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 1 2 7 - 3
72
C. Kauffmann et al. / Journal of Biotechnology 73 (1999) 71–74
enzymes to degrade or remove the compounds that cannot be treated satisfactorily by whole cell systems. For these reasons, the use of cell-free enzyme preparations for wastewater treatment has been suggested. The idea of using peroxidase, laccase and/or tyrosinase enzymes for the removal of phenols and aromatic amines from drinking and wastewater emerged in the 1980s and research in this field is still going on (Klibanov et al., 1980; Fiessinger et al., 1984; Shuttleworth and Bollag, 1986; Crecchio et al., 1995; Lee et al., 1996). After being oxidized the aromatic compounds will polymerize and form non-soluble compounds which can be removed by filtration or precipitation. A general conclusion reached from the work done with horseradish peroxidase (HRP) was that a microbial peroxidase would be needed in order to improve the economic feasibility of the method (Klibanov et al., 1983). Over the last decade much research has been carried out describing the fungal Coprinus cinereus peroxidase (CIP). The purification, crystallization and structural characterization of CIP were first reported by Morita et al. (1988). Andersen et al. (1991a,b) reported on kinetics and equilibria studies of CIP. Petersen et al. (1993) analyzed recombinant CIP expressed in Aspergillus oryzae. Veitch et al. (1994) compared wild-type, recombinant and three mutants of CIP. Tams and Welinder (1996) investigated denaturing and renaturing CIP. Smulevich et al. (1996) and Veitch et al. (1996) conducted detailed comparative studies of wild-type CIP and its Asp245-Asn (D245N) mutant. These and many other studies of CIP indicate its potential as the microbial peroxidase which has been sought after as an alternative to HRP for the treatment of phenolic wastewater. This article describes the removal of a number of phenols from water by treatment with CIP and H2O2.
mg of purpurgallin from pyrogallol in 20 s at pH 6.0 at 20°C (Sigma Chemical Company, 1984). All other chemicals were purchased from standard sources and were at least of reagent grade. The 10 phenols investigated were: phenol, 3-hydroxyphenol (resorcinol, 3-OH), 3-hydroxybenzoic acid (3COOH), 4-hydroxybenzoic acid (4-COOH), 2-methylphenol (o-cresol, 2-CH3), 3-methylphenol (m-cresol, 3-CH3), 2-chlorophenol (2-Cl), 4chlorophenol (4-Cl), 3-aminophenol (3-NH2), and 2,6-dichlorophenol (2-Cl,6-Cl). Concentrations of phenols were determined according to the original method of Emerson (1943) with the following modifications: to 1 ml of a phenol solution were added 0.20 ml 0.18 M NH3 and 1.13 M phosphate buffer pH 6.8 (less than 0.20 ml) until the pH of the solution was 7.9. To this was added 10 mM phosphate buffer pH 7.9 up to a volume of 1.40 ml. Then 0.20 ml 0.281% (w/v) 4-aminoantipyrine and 0.20 ml 1.13% (w/v) K3Fe(CN)6 were added. After 15 min for full colour development the absorbance at 500 nm was measured. This absorbance was proportional to the concentration of the examined phenols in the range 0–0.05 mg ml − 1. All samples were diluted to bring the phenol concentration into this range. Background samples were treated in a similar manner, but with water added instead of 4-aminoantipyrine.
2. Material and methods
3. Results and discussion
Wild-type CIP was cultured and provided by Novo Nordisk, Denmark. Activity was determined by measuring the formation of purpurgallin from pyrogallol. One unit would form 1.0
Immediately upon addition of peroxidase and H2O2 the investigated phenol solutions became coloured. The colour varied between milky white, yellow, orange, darkish brown and green depend-
2.1. Peroxidase treatment Phenols, H2O2 and CIP were mixed in 10 mM phosphate buffer to yield 0.1 mg ml − 1 phenol, 1 mM H2O2 and 1 U ml − 1 CIP. The mixtures were incubated in Erlenmeyer flasks for 3 h at 25°C in darkness. The precipitate formed was removed by centrifugation and the resultant clear supernatant was analyzed for the presence of residual phenols as described above.
C. Kauffmann et al. / Journal of Biotechnology 73 (1999) 71–74
73
ing on the phenol. Subsequently a precipitate formed, the amount of which also varied from phenol to phenol. Removal efficiencies at pH 6 and pH 7 are illustrated in Fig. 1. The investigated phenols, apart from resorcinol, could all be removed with high efficiencies (\90%) at one or both of the applied experimental conditions. Treatment with either peroxidase or H2O2 alone resulted in no appreciable phenol removal (data not shown).
Fig. 2. Percentage of phenol removed from solution as a function of pH (0.1 mg ml − 1 phenol, 1 mM H2O2 and 1 U ml − 1 CIP).
Fig. 1. Percentage of phenol removed from solution at pH 6 and 7 for phenols with different substituent groups (0.1 mg ml − 1 phenol, 1 mM H2O2 and 1 U ml − 1 CIP). *Experiments with 3-aminophenol were conducted at pH 5.5. Functional groups are explained in Section 2.
The effect of pH on the removal efficiency was investigated in the case of phenol (Fig. 2). The results showed a pH optimum around pH 8, which indicate that all or some of the removal efficiencies presented in Fig. 1 could be improved by increasing the pH. Further, by increasing the concentration of H2O2 from 1 to 1.5 mM the removal efficiency for phenol increased from 91 to 99% at pH 7 (data not shown). Some phenols, such as resorcinol, partly failed to precipitate after treatment with CIP. However, Klibanov et al. (1980) discovered in experiments with HRP that easily removed phenols (i.e. those that have high removal efficiencies) aid in the precipitation of other phenols. Real industrial wastewaters will generally contain a number of different pollutants. Hence, even if just a few of them are easily precipitated by peroxidase, they will facilitate the removal of the others by the enzyme and hydrogen peroxide. The main importance of the findings presented is that the microbial CIP can be used for the precipitation of phenols from aqueous solutions as efficiently as HRP (Klibanov et al., 1980). Further, the number of reports which have recently been published on wild-type and recombinant CIP and its mutants indicate the importance of this enzyme for phenol removal from industrial waste waters.
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C. Kauffmann et al. / Journal of Biotechnology 73 (1999) 71–74
References Andersen, M.B., Hsuanyu, Y., Welinder, K.G., Schneider, P., Dunford, H.B., 1991a. Kinetics and equilibria of cyanide binding to Coprinus cinereus peroxidase. Acta Chem. Scand. 45, 206 – 211. Andersen, M.B., Hsuanyu, Y., Welinder, K.G., Schneider, P., Dunford, H.B., 1991b. Spectral and kinetic properties of oxidized intermediates of Coprinus cinereus peroxidase. Acta Chem. Scand. 45, 1080–1086. Crecchio, C., Ruggiero, P., Pizzigallo, M.D.R., 1995. Polyphenoloxidases immobilization in organic gels: properties and applications in the detoxification of aromatic compounds. Biotechnol. Bioeng. 48, 585–591. Emerson, E., 1943. The condensation of aminoantipyrine. II. A new color test for phenolic compounds. J. Org. Chem. 8, 417 – 428. Fiessinger, F., Maloney, S.W., Manem, J., Mallevialle, J., 1984. Potential use of enzymes as catalysts in drinking water for the oxidation of taste causing substances. Aqua 2, 116 – 118. Klibanov, A.M., Alberti, B.N., Morris, E.D., Felshin, L.M., 1980. Enzymatic removal of toxic phenols and anilines from waste water. J. Appl. Biochem. 2, 414–421. Klibanov, A.M., Tu, T.M., Scott, K.P., 1983. Peroxidasecatalysed removal of phenols from coal-conversion waste waters. Science 221, 259–261. Lee, S.G., Hong, S.P., Sung, M.H., 1996. Removal and bioconversion of phenol in wastewater by a thermostable beta-tyrosinase. Enzyme Microb. Technol. 19, 374–377.
.
Morita, Y., Yamashita, H., Mikami, B., Iwamoto, H., Aibara, S., Terada, M., Minami, J., 1988. Purification, crystallization, and characterization of peroxidase from Coprinus cinereus. J. Biochem. 103, 693 – 699. Petersen, J.F.W., Tams, J.W., Vind, J., Svensson, A., Dalborge, H., Welinder, K.G., Larsen, S., 1993. Crystallization and X-ray diffraction analysis of recombinant Coprinus cinereus peroxidase. J. Mol. Biol. 232, 989 – 991. Shuttleworth, K.L., Bollag, J.M., 1986. Soluble and immobilized laccase as catalysts for the transformation of substituted phenols. Enzyme Microb. Technol. 8, 171 – 177. Sigma Chemical Company, 1984. Assay reagents and procedure; Prod. no. p-8125; Peroxidase. Sigma, St. Louis, MO. Smulevich, G., Neri, F., Marzocchi, M.P., Welinder, K.G., 1996. Versatility of heme coordination demonstrated in a fungal peroxidase: Absorption and resonance Raman studies of Coprinus cinereus peroxidase and the Asp245 Asn mutant at various pH values. Biochemistry 35, 10576 – 10585. Tams, J.W., Welinder, K.G., 1996. Unfolding and refolding of Coprinus cinereus peroxidase at high pH, in urea, and at high temperature. Effect of organic and ionic additives on these processes. Biochemistry 35, 7573 – 7579. The Merck Index, 1996. Merck, NJ. Veitch, N.C., Tams, J.W., Vind, J., Dalboge, H., Welinder, K.G., 1994. NMR studies of recombinant Coprinus peroxidase and three site-directed mutants: Implications for peroxidase substrate binding. Eur. J. Biochem. 222, 909 – 918. Veitch, N.C., Gao, Y., Welinder, K.G., 1996. The Asp245 Asn mutant of Coprinus cinereus peroxidase: Characterization by 1H-NMR spectroscopy and comparison with the wildtype enzyme. Biochemistry 35, 14370 – 14380.
Short communication
Enzymatic removal of phenols from aqueous solutions by Coprinus cinereus peroxidase and hydrogen peroxide Carl Kauffmann a,*, B.R. Petersen b, Morten J. Bjerrum c a
The Otto Warburg Center for Biotechnology in Agriculture, Faculty of Agriculture, The Hebrew Uni6ersity of Jerusalem, 76100 Reho6ot, Israel b No6o Nordisk A/S, No6o Alle, 2880 Bags6aerd, Denmark c Chemistry Department, The Royal Veterinary and Agricultural Uni6ersity, Thor6aldsens6ey; 40, 1871 Frederiksberg C, Denmark Received 20 October 1998; accepted 29 March 1999
Abstract The fungal enzyme Coprinus cinereus peroxidase (CIP) can be used for the removal of toxic phenols from water. After treating aqueous solutions of phenols with CIP and H2O2 the phenols polymerized and precipitated. The decrease in phenol concentration was investigated for 10 different phenols. At neutral pH, the investigated phenols were in general removed with high efficiency. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Bioremediation; Coprinus cinereus peroxidase; Environmental biotechnology; Phenols
1. Introduction Various phenols are inherent in wastewaters from a number of industries. Nearly all phenols are toxic and some are known to be human carcinogens. Therefore removal of phenols from industrial aqueous effluents is of great practical significance (Klibanov et al., 1980; The Merck Index, 1996). Removal and/or detoxification of pollutants can be done by physical, chemical or biological * Corresponding author. Tel.: +972-8-948-1973; fax: + 972-8-946-8785. E-mail address: [email protected] (C. Kauffmann)
means, the latter being of special interest because of its potential low cost and low adverse effect on the environment. Because of their biocidic nature most phenols cannot be satisfactorily degraded during conventional biological wastewater treatment, and the presence of phenols in high concentrations may irreversibly shock and damage live microbial systems. Enzymatic wastewater treatment opens a new avenue for the treatment of effluent streams containing hazardous or xenobiotic organic pollutants. The use of enzymes allows the degradation to be carried out under mild biological conditions, and the rationale is to use free or immobilized
0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 1 2 7 - 3
72
C. Kauffmann et al. / Journal of Biotechnology 73 (1999) 71–74
enzymes to degrade or remove the compounds that cannot be treated satisfactorily by whole cell systems. For these reasons, the use of cell-free enzyme preparations for wastewater treatment has been suggested. The idea of using peroxidase, laccase and/or tyrosinase enzymes for the removal of phenols and aromatic amines from drinking and wastewater emerged in the 1980s and research in this field is still going on (Klibanov et al., 1980; Fiessinger et al., 1984; Shuttleworth and Bollag, 1986; Crecchio et al., 1995; Lee et al., 1996). After being oxidized the aromatic compounds will polymerize and form non-soluble compounds which can be removed by filtration or precipitation. A general conclusion reached from the work done with horseradish peroxidase (HRP) was that a microbial peroxidase would be needed in order to improve the economic feasibility of the method (Klibanov et al., 1983). Over the last decade much research has been carried out describing the fungal Coprinus cinereus peroxidase (CIP). The purification, crystallization and structural characterization of CIP were first reported by Morita et al. (1988). Andersen et al. (1991a,b) reported on kinetics and equilibria studies of CIP. Petersen et al. (1993) analyzed recombinant CIP expressed in Aspergillus oryzae. Veitch et al. (1994) compared wild-type, recombinant and three mutants of CIP. Tams and Welinder (1996) investigated denaturing and renaturing CIP. Smulevich et al. (1996) and Veitch et al. (1996) conducted detailed comparative studies of wild-type CIP and its Asp245-Asn (D245N) mutant. These and many other studies of CIP indicate its potential as the microbial peroxidase which has been sought after as an alternative to HRP for the treatment of phenolic wastewater. This article describes the removal of a number of phenols from water by treatment with CIP and H2O2.
mg of purpurgallin from pyrogallol in 20 s at pH 6.0 at 20°C (Sigma Chemical Company, 1984). All other chemicals were purchased from standard sources and were at least of reagent grade. The 10 phenols investigated were: phenol, 3-hydroxyphenol (resorcinol, 3-OH), 3-hydroxybenzoic acid (3COOH), 4-hydroxybenzoic acid (4-COOH), 2-methylphenol (o-cresol, 2-CH3), 3-methylphenol (m-cresol, 3-CH3), 2-chlorophenol (2-Cl), 4chlorophenol (4-Cl), 3-aminophenol (3-NH2), and 2,6-dichlorophenol (2-Cl,6-Cl). Concentrations of phenols were determined according to the original method of Emerson (1943) with the following modifications: to 1 ml of a phenol solution were added 0.20 ml 0.18 M NH3 and 1.13 M phosphate buffer pH 6.8 (less than 0.20 ml) until the pH of the solution was 7.9. To this was added 10 mM phosphate buffer pH 7.9 up to a volume of 1.40 ml. Then 0.20 ml 0.281% (w/v) 4-aminoantipyrine and 0.20 ml 1.13% (w/v) K3Fe(CN)6 were added. After 15 min for full colour development the absorbance at 500 nm was measured. This absorbance was proportional to the concentration of the examined phenols in the range 0–0.05 mg ml − 1. All samples were diluted to bring the phenol concentration into this range. Background samples were treated in a similar manner, but with water added instead of 4-aminoantipyrine.
2. Material and methods
3. Results and discussion
Wild-type CIP was cultured and provided by Novo Nordisk, Denmark. Activity was determined by measuring the formation of purpurgallin from pyrogallol. One unit would form 1.0
Immediately upon addition of peroxidase and H2O2 the investigated phenol solutions became coloured. The colour varied between milky white, yellow, orange, darkish brown and green depend-
2.1. Peroxidase treatment Phenols, H2O2 and CIP were mixed in 10 mM phosphate buffer to yield 0.1 mg ml − 1 phenol, 1 mM H2O2 and 1 U ml − 1 CIP. The mixtures were incubated in Erlenmeyer flasks for 3 h at 25°C in darkness. The precipitate formed was removed by centrifugation and the resultant clear supernatant was analyzed for the presence of residual phenols as described above.
C. Kauffmann et al. / Journal of Biotechnology 73 (1999) 71–74
73
ing on the phenol. Subsequently a precipitate formed, the amount of which also varied from phenol to phenol. Removal efficiencies at pH 6 and pH 7 are illustrated in Fig. 1. The investigated phenols, apart from resorcinol, could all be removed with high efficiencies (\90%) at one or both of the applied experimental conditions. Treatment with either peroxidase or H2O2 alone resulted in no appreciable phenol removal (data not shown).
Fig. 2. Percentage of phenol removed from solution as a function of pH (0.1 mg ml − 1 phenol, 1 mM H2O2 and 1 U ml − 1 CIP).
Fig. 1. Percentage of phenol removed from solution at pH 6 and 7 for phenols with different substituent groups (0.1 mg ml − 1 phenol, 1 mM H2O2 and 1 U ml − 1 CIP). *Experiments with 3-aminophenol were conducted at pH 5.5. Functional groups are explained in Section 2.
The effect of pH on the removal efficiency was investigated in the case of phenol (Fig. 2). The results showed a pH optimum around pH 8, which indicate that all or some of the removal efficiencies presented in Fig. 1 could be improved by increasing the pH. Further, by increasing the concentration of H2O2 from 1 to 1.5 mM the removal efficiency for phenol increased from 91 to 99% at pH 7 (data not shown). Some phenols, such as resorcinol, partly failed to precipitate after treatment with CIP. However, Klibanov et al. (1980) discovered in experiments with HRP that easily removed phenols (i.e. those that have high removal efficiencies) aid in the precipitation of other phenols. Real industrial wastewaters will generally contain a number of different pollutants. Hence, even if just a few of them are easily precipitated by peroxidase, they will facilitate the removal of the others by the enzyme and hydrogen peroxide. The main importance of the findings presented is that the microbial CIP can be used for the precipitation of phenols from aqueous solutions as efficiently as HRP (Klibanov et al., 1980). Further, the number of reports which have recently been published on wild-type and recombinant CIP and its mutants indicate the importance of this enzyme for phenol removal from industrial waste waters.
74
C. Kauffmann et al. / Journal of Biotechnology 73 (1999) 71–74
References Andersen, M.B., Hsuanyu, Y., Welinder, K.G., Schneider, P., Dunford, H.B., 1991a. Kinetics and equilibria of cyanide binding to Coprinus cinereus peroxidase. Acta Chem. Scand. 45, 206 – 211. Andersen, M.B., Hsuanyu, Y., Welinder, K.G., Schneider, P., Dunford, H.B., 1991b. Spectral and kinetic properties of oxidized intermediates of Coprinus cinereus peroxidase. Acta Chem. Scand. 45, 1080–1086. Crecchio, C., Ruggiero, P., Pizzigallo, M.D.R., 1995. Polyphenoloxidases immobilization in organic gels: properties and applications in the detoxification of aromatic compounds. Biotechnol. Bioeng. 48, 585–591. Emerson, E., 1943. The condensation of aminoantipyrine. II. A new color test for phenolic compounds. J. Org. Chem. 8, 417 – 428. Fiessinger, F., Maloney, S.W., Manem, J., Mallevialle, J., 1984. Potential use of enzymes as catalysts in drinking water for the oxidation of taste causing substances. Aqua 2, 116 – 118. Klibanov, A.M., Alberti, B.N., Morris, E.D., Felshin, L.M., 1980. Enzymatic removal of toxic phenols and anilines from waste water. J. Appl. Biochem. 2, 414–421. Klibanov, A.M., Tu, T.M., Scott, K.P., 1983. Peroxidasecatalysed removal of phenols from coal-conversion waste waters. Science 221, 259–261. Lee, S.G., Hong, S.P., Sung, M.H., 1996. Removal and bioconversion of phenol in wastewater by a thermostable beta-tyrosinase. Enzyme Microb. Technol. 19, 374–377.
.
Morita, Y., Yamashita, H., Mikami, B., Iwamoto, H., Aibara, S., Terada, M., Minami, J., 1988. Purification, crystallization, and characterization of peroxidase from Coprinus cinereus. J. Biochem. 103, 693 – 699. Petersen, J.F.W., Tams, J.W., Vind, J., Svensson, A., Dalborge, H., Welinder, K.G., Larsen, S., 1993. Crystallization and X-ray diffraction analysis of recombinant Coprinus cinereus peroxidase. J. Mol. Biol. 232, 989 – 991. Shuttleworth, K.L., Bollag, J.M., 1986. Soluble and immobilized laccase as catalysts for the transformation of substituted phenols. Enzyme Microb. Technol. 8, 171 – 177. Sigma Chemical Company, 1984. Assay reagents and procedure; Prod. no. p-8125; Peroxidase. Sigma, St. Louis, MO. Smulevich, G., Neri, F., Marzocchi, M.P., Welinder, K.G., 1996. Versatility of heme coordination demonstrated in a fungal peroxidase: Absorption and resonance Raman studies of Coprinus cinereus peroxidase and the Asp245 Asn mutant at various pH values. Biochemistry 35, 10576 – 10585. Tams, J.W., Welinder, K.G., 1996. Unfolding and refolding of Coprinus cinereus peroxidase at high pH, in urea, and at high temperature. Effect of organic and ionic additives on these processes. Biochemistry 35, 7573 – 7579. The Merck Index, 1996. Merck, NJ. Veitch, N.C., Tams, J.W., Vind, J., Dalboge, H., Welinder, K.G., 1994. NMR studies of recombinant Coprinus peroxidase and three site-directed mutants: Implications for peroxidase substrate binding. Eur. J. Biochem. 222, 909 – 918. Veitch, N.C., Gao, Y., Welinder, K.G., 1996. The Asp245 Asn mutant of Coprinus cinereus peroxidase: Characterization by 1H-NMR spectroscopy and comparison with the wildtype enzyme. Biochemistry 35, 14370 – 14380.