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Color removal from Kraft effluent by lignin peroxidase and horseradish ... horseradish peroxidase on CNBr-Sepharose 4B improved the decolorization by factor.

Biotechnology Letters Vol 13 No 3 Received 9th July

577-532

(1991)

DECOLORIZATION OF KRAFT EFFLUENT BY FREE AND IMMOBILIZED LIGNIN PEROXlDASES AND HORSERADISH PEROXlDASE Irene Ferrer#, Marcia Dezotti& and Nelson Durdn * Facultad de Qufmica, Pontificia Universidad Cat61ica de Chile, Casilla 6177, Santiago, Chile#, and Instituto de Qufmica, Biological Chemistry Laboratory, Universidade Estadual de Campinas, C.P. 6154, Campinas, CEP 13081 S.P., Brazil& ABSTRACT Color removal from Kraft effluent by lignin peroxidase and horseradish peroxidase was compared. Free lignin peroxidase and horseradish peroxidase removed color from kraft effluent. Immobilization of lignin peroxidase type III, lyophilized fungal culture and horseradish peroxidase on CNBr-Sepharose 4B improved the decolorization by factor of 2.9, 4.5 and 2.6, respectively in 48 h. Lignin peroxidase type I was effective only in the immobilized form in decolorization. In general, the immobilized form all the studied systems exhibited an average value around of 30% polymer consumption and very little of depolymerization. Lignin peroxidases and lyophilized fungal culture were shown to have considerable potential for treating Kraft effluents. INTRODUCTION The major phenolic contaminants in rivers are produced by pulp and paper industries, principally in the alkaline extraction stage of wood-pulp bleaching which accounts for over 50% of the color load. Conventional treatment methods, such as aerated lagoons and activated sludge plants, are ineffective in removing this color or are extremely expensive. Recently the potential uses of peroxidases were extended to removal of toxic wastes and they have been shown in laboratories to efficiently remove carcinogenic aromatic amines from aqueous industrial effluents (Klibanov and Morris, 1981) and low molecular weight color bodies from mill effluents (Paice and Jurasek, 1984;Jurasek and Paice, 1986; Hopkins, 1984).Despite these uses, the industrial application of peroxidases has been limited, mainly because of their relative instability, high isolation and purification costs, and the difficulty in recovering active enzyme after completion of the catalytic process (Kennedy and Cabral, 1987; Hakulin, 1987). Laccase and peroxidases oxidize phenolic compounds to aryloxy radicals, which spontaneously polymerize to form insoluble complexes which can be removed by precipitation, filtration or centrifugation (Davis and Burns, 1990). Lignin peroxidases are currently under study in the treatment of to Kraft effluent, but no data were discussed in term of its efficiency or a more detailed experiments(FarreTl, 1987; Aust et al., 1986). It have been shown that aeration in the presence of laccase removes soluble phenolic compounds and thereby color and toxicity from waste water from the pulp and paper industry (Forss et al., 1989). It is generally accepted that in order to increase the potential for the use of enzymes as a wastewater treatment method, it is necessary to immobilize the enzyme so that it is biochemically stabilized and reusable. Phenoloxidase from Trametes versicolor, an extracellular peroxidase from Inonotus radiatus, was immobilized on glass beads (Lobarsenski and Paszczynski, 1985), Laccase from Rhizoctonis praticola was immobilized by covalent coupling to Celite (Shuttleworth and Bollag, 1986) and lignin peroxidase M-2 from Phanerochate chrysospodum was reported to be immobilized in CNBr-Sepharose 4B, but no experimental results were discussed (Paszczynski et al., 1986; Henry et al., 1974). Horseradish peroxidase was immobilized in a similar manner (Pharmacia, 1986). Immobilization of lignin-modifying enzymes for use in organic solvents was reported (Huettermanns et al., 1988). Soluble laccase and horseradish peroxidase removed color ~To whom correspondence shoul be addressed.

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from pulp mill effluent, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase and the peroxidase in alginate beads improved the decolorization by several folds. Co-polymerization of laccase and horseradish peroxidase with L-tyrosine gave an insoluble polymer with enzyme activity. Entrapment of the co-polymer in gel beads further increased the efficiency of decolorization in the pulp mill effluent (Davis and Burns, 1990). Chloroperoxidase purified from Caldariomyces fumago was covalently bound to aminopropyl glass by using a modification of an established method (Kadima and Pickard, 1990). Very recently~ lignin peroxidase partially purified from P. chrysosporium was immobilized on glass beads and in activated agarose (Fawer et al., 1 991 ). In all cases a more stable enzyme was obtained. Since a drawback to use lignin peroxidase rather than fungi in effluent treatment is the cost of the enzyme, the operational stability of the catalyst is of paramount importance. We report here the effect of a simple immobilization method for enzymatic removal of colour from the Kraft effluent by lignin peroxidases from C. sitophila (Durdn et al., 1987). MATERIAL AND METHODS Culture conditions. The strain of Chrysonil/a s/toph/la (TFB-27441) and culture conditions used for production of the enzyme were described previously (Dur~n et al., 1987). Lignin peroxidases purifications. From protein elution three fractions were isolated which are described in TABLE 1 and their kinetic characteristics in TABLE 2. TABLE 1. GENERAL CHARACTERISTICS OF LIGNIN PEROXIDASE FROM C, SlTOPHILA (TFB-27441 STRAIN) LIGNIN PEROXIDASE MOL.WEIGHT (DALTON9)

pl

CARBOHYDRATE OPTIMUM CONTENT(%) pH temp. ~

LIG-I LIG-II LIG-III

9.05 6.74 4.50

25.7 14.8 17.4

68,000 50,200 47.500

3.0 5.0 4.0

28 28 28

Enzyme assays. Enzyme activity was measured by UV-spectroscopy of veratryl aldehyde (~l 310 nm) formed by the oxidation of veratryl alcohol at pH 2.5 (Dur,'1n et al., 1987). Immobilization of lignin and horseradish peroxidases, and filtered culture (Roy et al., 1989). One gram of CNBr-Sephadex 4B (SIGMA) was washed and reswelled on a sintered glass filter using 1 mM HCI for 1 h. Ten mg of purified enzyme or lyophilized fungal TABLE 2. KINETIC CONSTANTS OF LIGNIN PEROXIDASES FROM C. SITOPHILA (TFB-27441 STRAIN) CULTURE (a) LIGNINPEROXIDASES VERATRYLALDEHYDE KM app Vmax mM nmol.mim'lmL"1

HYDROGENPEROXIDE KM app Vmax uM nmol. mira"1 mL"1

LIG-I LIG-II LIG-III

50 37 33

0.37 0.30 0.12

9.09 9.30 2.50

6.67 7.20 4.80

a) The kinetic constant were calculated from Lineweaver-Burk plots. culture was taken in NaHCO3 buffer (10 mM, pH 8.3) containing NaCI (0.5 mM), mixed with gel suspension and then kept overnight at 4~ For blocking the remaining active groups, the gel suspension was transferred to blocking agent 0.2 M glycine, pH 8.0

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and to remove excess adsorbed protein, the gel was washed alternately three or four times w i t h high coupling buffer (pH 8.3) and Iow-pH Na-acetate buffer (4.2), each containing 0.5 M NaCI. This ensures that no free ligand remained ionicaHy bound to the immobilized ligands. T h e gels thus prepared were kept for further use. Immobilized enzymes were stored at 4 ~ w i t h 0 . 0 2 % sodium azide as antimicrobial agent. Kraft effluent treatment. The Kraft liquor (El) was diluted, filtered and the pHadjusted to 5.0 w i t h an absorbance of 0.5 (1,894 CU). To 10 ml of effluent, an adequated mass of immobilized enzyme complexes and 5 mM hydrogen peroxide at 2 5 ~ was added. Color removal by enzymes was measured in duplicate in 2 ml effluent previously centrifuged for 2 minutes at 1 5 , 0 0 0 rpm in a Eppendorf Microcentrifuge by monitoring the absorption at 465 nm. Molecular weight distribution. 0.5 ml of the clean effluent supernatant was applied to a 60 x 1.5 cm Sephadex G-50 column equilibrated with NaOH-LiCI 0.1 N, 2.5 ml were collected (flow rate of 2 min 15 s/ml) and the chlorolignins were determined at 2 8 0 rim.

RESULTS AND DISCUSSION. The percentage of activity retained on immobilization for each system is shown in TABLE 3. TABLF 3. RETENTJON OF LtGNIN PEROXJDASEACTIVITY FOLLOWJNG IMMOBI[.fZATION ON CNBr-ACTIVATED SEPHAROSE 4B. ENZYME

THEORETICALIMMOB. UNITS BY DRIEDGEL(mg) X 103

RETAINED UNITS RETENTION BY DRIEDGEL(mg) (%) X IO 3

HRP-Sepharose LIG-I-Sepharose LIG-III-Sepharose Lyophilizedfungal culture-Shepharose

1.0 8.1 79.0 26.0

0.38 0.16 6.3 0.25

38 2 8 1

No optimization to enhance the retention of the activity in the support was attempted. The best activity retention was w i t h HRP and then with LIG-III. Unfortunately the a m o u n t of LIG-II available for these experiments was insuficient for immobilization. Aditional studies are actually in progress. TABLE 4 shows the percentage of effluent decolorization obtained by the free and the immobilized forms on Sepharose 4B activated by CNBr, TABLE 4. PERCENTAGE OF DECOLORIZATION OF KRAFT EFFLUENT (a) EFFLUENTAND ENZYMES HRP (0.06 U) HRP-Sepharose(0.06 U) LIG-I (0.03 U) LIG-I-Sepharose(0.03 U) LIG-III (0.10 U) LIG-III-Sepharose(0.10 U) Lyophilizedfungal cuJture (0.03 U) Lyophiiizedfungal culture (0.03 U| -Sepharose Hydrogen peroxide (5 raM)

0 0 0 0 0 0 0 0 0

TIME (h) 24 14.4 26,6 2,0 16.9 10.2 21.6 5.4 27.2

48 20.2 53.5 2.0 16.0 13.3 38.4 6.4 29.3

72 25.0 61.3 2.0 2.0 30.7 45.7 2.0 11.4

0

2.0

2.0

2.0

a) 10 ml effluent pH 5, hydrogen peroxide 5 mM at 2 5 ~ LIG-I and in lyophilized culture at 72 h the color was recovered. With HRP, a 2.5 fold increase of decolorization w i t h the immobilized enzyme was obtained. LIG-I did not decolorized the effluent at the concentration studied, but significant decolorization in

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