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Vol. 178, No. 3, 1991 August
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1092-1098
OXIDATIVE DEGRADATION OF HIGH MOLECULAR WEIGHT CHLOROLIGNIN BY MANGANESE PEROXIDASE OF PHANEROCHAETE CHRYSOSPORIUM Richard Lackner, Ewald Srebotnik’ and Kurt Messner Abteilung Mykologie, Institut fur Biochemische Technologie und Mikrobiologie, Technische Universitat Wien, Getreidemarkt 9, A-1060 Wien, Austria Received
June
24,
1991
Phanerochaete chrysospotium was able to degrade high molecular weight chlorolignins (IvJ >30,000) from bleach plant effluents, although a direct contact between ligninolytic enzymes and chlorolignin was prevented by a dialysis tubing. In the absence of the enzymes, Mn 3+ depolymerized chlorolignin when complexed with lactate causing the color, chemical oxygen demand (COD) and dry weight to decrease by 80%! 60% and 40%, respectively. Manganese peroxidase effectively catalyzed the depolymerization of chlorolignin in the prance of Mn2+ and H202. It can be concluded from these results that manganese peroxidase plays the major role in the initial breakdown and decolorization of high molecular weight chlorolignin in bleach plant effluents by P. chrysosporiumin v&o. o1991Academic~re~~,~nc. SUMMARY:
Pulp is bleached by chlorination of the residual lignin and subsequent alkali extraction. The effluent of the first alkali stage (El) contains numerous chlorinated and oxidized lignin related compounds (chlorolignins) of which the high molecular weight fraction can not be degraded in aerated lagoons. White rot fungi are unique in their ability to depolymerize and metabolize lignin and Phanerochaetechrysosporiumsecretes lignin peroxidases and manganese peroxidases as the major components of its ligninolytic system (l-3). Both enzymes are believed to play important roles in lignin degradation (4) and exhibit a broad spectrum of oxidative biodegradation potential due to single-electron oxidation (5-6). The degradation of several low moIecular weight model compounds by lignin peroxidase and manganese peroxidase has been demonstrated (7), but these enzymes have never been shown to catalyze the depolymerization of native lignin in tivo. Only recently has the depolymerization of a synthetic lignin (DHP, dehydropolymerizate) by lignin peroxidase (8) and manganese peroxidase (9) been demonstrated. However, it is not known how closely this soluble model substance of relatively low molecular weight (M, >2000) resembles native lignin in vivo. Depolymerization seems to be a key reaction not only for the degradation of native lignin but also for the degradation of high molecular weight chlorolignin, leading to low molecular weight products which can be further metabolized intracellularly. Chang et al. (10) have used P. chtysosporium, immobilized in a rotating biological contactor, to degrade chlorolignins in * To whom correspondence should be addressed.
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bleach plant effluents and Pellinen et al. (11) have shown dechlorination, decolorization, and degradation of high molecular weight chlorolignins in that system. Messner et al. (12) obtained about 80% decolorization in 24 hours using a trickling filter reactor with P. chrysosporium immobilized on foam. However, very little is known about the mechanism of chlorolignin degradation by white rot fungi and so far, the involvement of either lignin peroxidase or manganese peroxidase has not been demonstrated. Mn3+, stabilized with organic acids, has a high redox potential and oxidizes a variety of organic lignin model compounds (5, 13-16) and is produced when manganese peroxidase of P. chrysosporium catalyzes the oxidation of Mn2+ to Mn3+ by H,O, (2, 13). In the present study we demonstrate for the tirst time the involvement of Mn3+ and manganese peroxidase in the biodegradation of chlorolignin by P. chrysosporium. MATERIALS AND METHODS Preparation of hiph molecular weight chlorolienin: The chlorinated, first alkali stage effluent (El) from a Austrian sulfite plant containing 300-500 mg/l AOX was fractionated by diafiltration (17) against water, using a 30 kDa cut-off ultrafiltration membrane (Amicon YM-30). 40% of the total color in the effluent was due to the high molecular weight fraction M, > 30,000. 50 ml portions were packed into dialysis tubings (Servapore, 10-15 kDa cut-off) and incubated with chelated Mn3+, manganese peroxidase/Mn2+/H202 or P. chrysosporium cultures. Degradation of chlorolienin by P. chyvsos~orium: The white-rot fungus P. chryosporium BKM F-1767 (ATCC 24725) was grown immobilized on 1 cm3 polyurethane foam cubes in 1 liter Erlenmeyer flasks containing 250 ml of a defined medium modified from Kirk et al. (18) with 1.25 g/l diammonium tartrate and 10 mM sodium phthalate at pH 4.5 on a rotary shaker (120 rpm) at 39°C. Beginning on day 5, the culture fluid was harvested every 24 hours and replaced by fresh medium containing 0.22 g/l diammonium tartrate and high molecular weight chlorolignin (HMW chlorolignin) instead of phthalate buffer. In other experiments, the HMW chlorolignin was packed into dialysis tubing and incubated with ligninolytic cultures of the fungus for several days. Once a day, the dialysis tubing was changed and small samples were taken for analysis. Controls were run (detection of enzyme activities and protein concentrations in and outside the tubing) to ensure that the permeability of the tubing was not changed due to enzymatic degradation. Degradation of chlorolienin with Mn3+: 50 ml HMW chlorolignin was packed into a dialysis tubing and incubated in 1 liter Erlenmeyer flasks with 250 ml of 4 mM manganese(III)acetate dihydrate (Aldrich), chelated with 0.5 M sodium lactate, pH 4.5, on a rotary shaker at 80 rpm and 39°C. The manganese lactate medium was renewed every 12-15 hours. Controls were run using sodium lactate buffer without Mn3+. Degradation of chlorolienin with maneanese oeroxidase: Depolymerization was carried out, as described above for Mr?‘, with 500 U/l manganese peroxidase and 0.5 mM MnSO, dissolved in 0.5 M sodium lactate, pH 4.5. H202 was continuously added at a rate of 7 pmol/min.l. Controls were run omitting either manganese peroxidase, Mn2+ or H202, or using 0.5 mM Mn3+ instead of Mn’+, manganese peroxidase, and H202. Manganese peroxidase was isolated from liquid cultures of P. chrysosporium lacking lignin peroxidase activity and purified by FPLC on a Mono Q column (Pharmacia-LKB). Analvsis of degradation oroducts: Size exclusion chromatography of chlorolignin was performed by HPLC using a Bio-Bad model 400 chromatograph and a TSK G3OOOSW column (7.5 x 600 mm, Pharmacia-LKB) with 0.03 M sodium acetate buffer, pH 6.7 and 0.5 g/l polyethylene glycol as the mobile phase at a flow rate of 1 ml/min. Bleach plant effluent (El), fractionated by ultra-filtration as well as blue dextrane and 4-chlorophenol were used to calibrate the column (Fig 2A). The color of the effluent was measured spectrophotometrically at 465 nm according to Eaton et al. (19) and expressed as color units (CU/l). The chemical oxygen demand (COD) was measured using a commercial assay kit (Dr. Lange, Austria) according to the supplier. 1093
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For weight loss determination the degraded HMW chlorolignin was dialyzed against water, concentrated by evaporation and dried at 60°C as well as at 110°C. Electronhoresis and immunoblottinp: The culture filtrates were concentrated 25 to 75-fold prior to electrophoresis (Amicon Centricon 10) and protein concentrations were measured using Bio-Rad’s Coomassie blue microassay. Polyacrylamide gel electrophoretic analysis (20) and immunoblotting (Western blotting) of ligninase proteins (lignin peroxidase and manganese peroxidase) were performed as described elsewhere (21). Enzvme assavs: Lignin peroxidase activity in culture fluids was measured spectrophotometrically using veratrylalkohol as a substrate (18) and manganese peroxidase was assayed with 2,bdimethoxyphenol according to Paszczynski et al. (3). One unit (II) of activity was defined as the oxidation of 1 pmol substrate per minute at 25°C for both enzymes. Manganese peroxidase was extracted with 0.5 M NaCl after washing of the immobilized fungus several times with destilled water. RESULTS
AND DISCUSSION
Fig. 1A shows lignin peroxidase and manganese peroxidase production, and degradation of high molecular weight chlorolignin in a semicontinuous culture of Phunerochaete chlysosporium. From day 5 to day 25, the culture fluid was decanted and replaced with fresh medium every day. The data, therefore, represent what was produced or degraded within 24 hours. Manganese peroxidase was measured directly in the culture fluid containing chlorolignin, whereas lignin peroxidase, for known methodical reasons, could be measured only in parallel cultures without chlorolignin. Interestingly, color decrease was still high after 25 days of cultivation (35%) although ligninolytic activity decreased to almost zero after 17 days. To ensure the absence of ligninase protein (lignin peroxidase and manganese peroxidase), the culture filtrate was concentrated 75-fold and separated by electrophoresis. No protein could be detected in 21 day old cultures (Fig. 1B) and immunoblotting did not show significant amounts of ligninase proteins (Fig. 1C). However, considerable amounts of manganese peroxidase (corresponding to
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(+) Ligninperoxidase production and (s) manganese production (U/l) and (*) of chlorolignin (910colour decrease)in semicontinuous liquid cultures of P. chysosporium [A]. Elecrophoretic separation [B] and immunoblotting [C] of the culture fluids after 5 days (lanes a, concentrated 25-fold) and 21 days (lanes b, concentrated75-fold) of cultivation. Lanes c: ligninase standard (control). decolonzahon
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50-100 U/l) but no lignin peroxidase was readily extractable from the mycelium with 0.5 M NaCl. This is in agreement with the finding of Paszczynski et al. (3) that manganese peroxidase is at least partly associated closely with the mycelium. Therefore, it seemed likely that mycelium bound manganese peroxidase might play an important role in bleach plant effluent degradation. To show this, we performed subsequent experiments during which the high molecular weight chlorolignin was not added directly to the cultures but packed into dialysis tubings and incubated with the fungus for several days. Decolorization of the effluent was observed (Fig. 3A) and the chlorolignin was depolymerized (Fig. 2B) although the fungal enzymes could not directly react
I3
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Eig-2. Gel chromatography of high molecularweightchloroligninin dialysistuhingsbefore (-) and after (---) treatmentwith P. chtysosporizun for 3 days[B], Mn3+for 2 days [Cl, and manganese peroxidasefor 3 days[D]. GraphA: calibrationof thecolumnwith bluedextran(l), 4-chlorophenol(5) andfractionatedbleachplant effluentwith m, > 30,000(2), 1000-5000(3), and < 1000(4).
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with the effluent due to the dialysis membrane. No manganese peroxidase could be detected inside the tubing whereas high activities were measured in the culture fluid outside the tubing. Due to diffusion limitation, a slower degradation rate than without dialysis tubing was observed. These results indicated that a low molecular weight agent degraded the polymeric bleach plant effluent. The small, diffusible manganese-(III)-complex, generated by manganese peroxidase, could be this reactive molecule. Using Mn3+-lactate as a biomimetic model for enzymatically generated Mn3+ (13), the oxidation of polymeric chlorolignin was also performed in dialysis tubings and a decolorization of about 80% was reached after 4-5 days of incubation (Fig. 3A). The manganese solution outside the tubing was changed every 12-15 hours because of the reported half life time of about 10 hours for Mn3+ due to hydrolyzation and disproportionation to Mn4’ (MnO*) and Mn’+ (13). After dialysis against water to remove interfering lactate and low molecular weight degradation products, a significant decrease in COD (60%) and dry weight (40%) could be measured (Table 1). The gel permeation elution profile was altered significantly and the adsorption at 280 nm (Fig. 2C) as well as at 465nm (not shown) decreased. Color decrease due to oxidation of the chromophores was twice as high as weight loss because only depolymerization products with molecular weights below 10,000-15,000 were removed by dialysis. In subsequent experiments, manganese peroxidase was used for Mn3’ generation from Mn2+ under optimum conditions for the enzyme (2). As the kinetics of chlorolignin oxidation by Mn3+ is not known, the rate of H202 addition had to be adjusted and 7 pmol/l.min at 37°C was found to be the optimum. The H,Oz was completely consumed and enzyme inactivation was low (15%/day) under these conditions. Degradation occured through the dialysis membrane indicating that a direct contact between enzymes and substrate (chlorolignin) was not necessary. Decolorization (Fig. 3A), weight loss (Table 1) and gel permeation profile (Fig. 2D) were similar to the enzyme-free Mn’+ -system, but COD decrease was lower, probably due to the
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.--.-.
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Fig. 3. Comparison of chlorolignin decolorization in time by P. chrysosporium in vivo,
manganese peroxidase, and Mn3+ [A]. Proposed mechanism of chlorolignin depolymerization in the dialysis tubing system [B]. 1096
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Table l.Decolorization, COD decrease, and weight loss of high molecular weight chlorolignin in dialysis tubings after treatment with Mn3+ and manganese peroxidase in time DAYS
Mn-(III)-COMPLEX 0 1 2 3 5 MANGANESE 0 1 2 3 4 5
COLOR m/1
%
COD w/l
%
WEIGHT w/l
%
563 478 388 304 123
0 15 31 46 78
79 61 38 35 31
0 23 51 55 61
326 300 280 244 186
0 8 14 25 43
PREOXIDASE 626 538 400 238 194 175
0 14 36 62 69 72
53 46 44 39 36 34
0 12 17 26 31 36
373 347 328 298 265 231
0 7 12 20 29 38
different manganese concentrations and, therefore, redox potentials in the two systems. No degradation was observed when manganese peroxidase, Mn2+, or H202 was omitted. Furthermore, controls with 0.5 mM Mn3+ did not show any significant decolorization, indicating that Mn 3+ had been continuously regenerated by the enzyme. The rate of degradation was much higher with Mn3+ and manganese peroxidase in vitro than with P. chrysosporium in tivo in all experiments, indicating that manganese peroxidase together with H202 generating enzymes is solely responsible for the initial breakdown of high molecular weight chlorolignin in bleach plant effluents by P. chrysosporium in vivo. The conclusions which may be drawn from the results are summarized in Fig. 3B. Mn3+, generated by extracellular manganese peroxidase of the fungus, freely diffuses into the dialysis tubing to react with chlorolignin. Low molecular weight degradation products can then diffuse out of the tubing to be metabolized by the fungus and Mn3+ is regenerated from Mn*+. Recently, Hammel and Moer (8) have demonstrated the depolymerization of dehydropolymerizate (DHP) by lignin peroxidase and Wariishi et al. (9) have very recently published a paper showing that manganese peroxidase can also depolymerize DHP. However, lignin peroxidase was not necessary for depolymerization of the chlorolignins in our experiments. Although the chemical properties of chlorolignin are very different from DHP and native lignin, our results confirmed the significance of manganese peroxidase in the initial breakdown of polymeric substrates. This work was supported by the “Forschungsforderungsfonds gewerblichen Wirtschaft” . Acknowledgment:
REFERENCES
2. 1. Tien, M., and Kirk, T.K. (1983) Science 221, 661-663. Glenn, J.K., and Gold, M.H. (1985) Arch. Biochem. Biophys. 242, 329-341. 1097
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3. Paszczynski, A., Huynh, V.-B., and Crawford, R.L. (1986) Arch. B&hem. Biophys. 244, 750-765. 4. Kirk, T.K. , and Farrell, R.L. (1987) Annu. Rev. Microbial. 41, 465-505. 5. Kersten, P.J., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612. 6. Harvey, P.J., Schoemaker, H.E., Bowen, R.M., and Palmer J.M. (1985) FEBS I.&t. 183, 13-16. 7. Higuchi, T. (1990) Wood Sci. Technol. 24, 23-63. 8. Hammel, K.G., and Moer, M.A. (1991) Enz. Microb. Technol.13, 15-18. 9. Wariishi, H., Valli, K., and Gold, M.H. (1991) Biochem. Biophys. Res. Comm. 176, 269-275. 10. Chang, H.-M., Joyce, T.W., Kirk, T.K., and Huynh, V.-B. (1984) US Patent No. 4554075 (Assignee: North Carolina State University, NC). 11. Pellinen, J., Joyce, T.W., and Chang, H.-M. (1988) Tappi J. 71, 191-194. 12. Messner, K., Ertler, G., Jaklin-Farther, S., Boskovsky, P., Regensberger, U. and Blaha, A. (1990) In Biotechnology in Pulp and Paper Manufacture (T.K. Kirk, and H.-M. Chang, Eds) pp. 245-253, Butterworth-Heinemann, Stoneham. 13. Glenn, J.K. , Akileswaran, L., and Gold, M.H. (1986) Arch. Biochem. Biophys. 251, 88-696. 14. Forrester, I.T., Grabski, A.C., Burgess, R.R., and L&ham G.F. (1988) B&hem. Biophys. Res. Comm. 157, 992-999. 15. Hammel, K.E., Tradone, P.J. Moen, M. A., and Price, L.A. (1989) Arch. Biochem. Biophys. 270, 404-409. 16. Waters, W.A., and Littlers, J.S. (196%) In Oxidation in Organic Chemistry (K. B. Wiberg, Ed.), Vol. 5A, pp. 185-241, Academic Press, New York. 17. Hardell, H.-L., and de Sousa, F. (1977) Svensk Papperstidning 4, 110-120. 18. Kirk, T.K. , Croat-t , S., Tien, M., Murtaugh, K., and Farrell, R. (1986) Enz. Microb. Technol. 8, 27-32. 19. Eaton, D.C., Chang, H.-M., and Kirk, T.K. (1980) Tappi J. 63, 103-106. 20. Laemmli, U.K. (1970) Nature 227, 680-685. 21. Srebotnik, E. Messner, K., and Foisner, R. (1988) Appl. Environ. Microbial. 54, 2608-2614. 22. Blanchette, R.A. (1984) Phytopathol. 74, 725-730. 23. Kern, H.W. (1989) Appl. Microbial. Biotechnol. 32, 223-234.
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OXIDATIVE DEGRADATION OF HIGH MOLECULAR WEIGHT CHLOROLIGNIN BY MANGANESE PEROXIDASE OF PHANEROCHAETE CHRYSOSPORIUM Richard Lackner, Ewald Srebotnik’ and Kurt Messner Abteilung Mykologie, Institut fur Biochemische Technologie und Mikrobiologie, Technische Universitat Wien, Getreidemarkt 9, A-1060 Wien, Austria Received
June
24,
1991
Phanerochaete chrysospotium was able to degrade high molecular weight chlorolignins (IvJ >30,000) from bleach plant effluents, although a direct contact between ligninolytic enzymes and chlorolignin was prevented by a dialysis tubing. In the absence of the enzymes, Mn 3+ depolymerized chlorolignin when complexed with lactate causing the color, chemical oxygen demand (COD) and dry weight to decrease by 80%! 60% and 40%, respectively. Manganese peroxidase effectively catalyzed the depolymerization of chlorolignin in the prance of Mn2+ and H202. It can be concluded from these results that manganese peroxidase plays the major role in the initial breakdown and decolorization of high molecular weight chlorolignin in bleach plant effluents by P. chrysosporiumin v&o. o1991Academic~re~~,~nc. SUMMARY:
Pulp is bleached by chlorination of the residual lignin and subsequent alkali extraction. The effluent of the first alkali stage (El) contains numerous chlorinated and oxidized lignin related compounds (chlorolignins) of which the high molecular weight fraction can not be degraded in aerated lagoons. White rot fungi are unique in their ability to depolymerize and metabolize lignin and Phanerochaetechrysosporiumsecretes lignin peroxidases and manganese peroxidases as the major components of its ligninolytic system (l-3). Both enzymes are believed to play important roles in lignin degradation (4) and exhibit a broad spectrum of oxidative biodegradation potential due to single-electron oxidation (5-6). The degradation of several low moIecular weight model compounds by lignin peroxidase and manganese peroxidase has been demonstrated (7), but these enzymes have never been shown to catalyze the depolymerization of native lignin in tivo. Only recently has the depolymerization of a synthetic lignin (DHP, dehydropolymerizate) by lignin peroxidase (8) and manganese peroxidase (9) been demonstrated. However, it is not known how closely this soluble model substance of relatively low molecular weight (M, >2000) resembles native lignin in vivo. Depolymerization seems to be a key reaction not only for the degradation of native lignin but also for the degradation of high molecular weight chlorolignin, leading to low molecular weight products which can be further metabolized intracellularly. Chang et al. (10) have used P. chtysosporium, immobilized in a rotating biological contactor, to degrade chlorolignins in * To whom correspondence should be addressed.
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bleach plant effluents and Pellinen et al. (11) have shown dechlorination, decolorization, and degradation of high molecular weight chlorolignins in that system. Messner et al. (12) obtained about 80% decolorization in 24 hours using a trickling filter reactor with P. chrysosporium immobilized on foam. However, very little is known about the mechanism of chlorolignin degradation by white rot fungi and so far, the involvement of either lignin peroxidase or manganese peroxidase has not been demonstrated. Mn3+, stabilized with organic acids, has a high redox potential and oxidizes a variety of organic lignin model compounds (5, 13-16) and is produced when manganese peroxidase of P. chrysosporium catalyzes the oxidation of Mn2+ to Mn3+ by H,O, (2, 13). In the present study we demonstrate for the tirst time the involvement of Mn3+ and manganese peroxidase in the biodegradation of chlorolignin by P. chrysosporium. MATERIALS AND METHODS Preparation of hiph molecular weight chlorolienin: The chlorinated, first alkali stage effluent (El) from a Austrian sulfite plant containing 300-500 mg/l AOX was fractionated by diafiltration (17) against water, using a 30 kDa cut-off ultrafiltration membrane (Amicon YM-30). 40% of the total color in the effluent was due to the high molecular weight fraction M, > 30,000. 50 ml portions were packed into dialysis tubings (Servapore, 10-15 kDa cut-off) and incubated with chelated Mn3+, manganese peroxidase/Mn2+/H202 or P. chrysosporium cultures. Degradation of chlorolienin by P. chyvsos~orium: The white-rot fungus P. chryosporium BKM F-1767 (ATCC 24725) was grown immobilized on 1 cm3 polyurethane foam cubes in 1 liter Erlenmeyer flasks containing 250 ml of a defined medium modified from Kirk et al. (18) with 1.25 g/l diammonium tartrate and 10 mM sodium phthalate at pH 4.5 on a rotary shaker (120 rpm) at 39°C. Beginning on day 5, the culture fluid was harvested every 24 hours and replaced by fresh medium containing 0.22 g/l diammonium tartrate and high molecular weight chlorolignin (HMW chlorolignin) instead of phthalate buffer. In other experiments, the HMW chlorolignin was packed into dialysis tubing and incubated with ligninolytic cultures of the fungus for several days. Once a day, the dialysis tubing was changed and small samples were taken for analysis. Controls were run (detection of enzyme activities and protein concentrations in and outside the tubing) to ensure that the permeability of the tubing was not changed due to enzymatic degradation. Degradation of chlorolienin with Mn3+: 50 ml HMW chlorolignin was packed into a dialysis tubing and incubated in 1 liter Erlenmeyer flasks with 250 ml of 4 mM manganese(III)acetate dihydrate (Aldrich), chelated with 0.5 M sodium lactate, pH 4.5, on a rotary shaker at 80 rpm and 39°C. The manganese lactate medium was renewed every 12-15 hours. Controls were run using sodium lactate buffer without Mn3+. Degradation of chlorolienin with maneanese oeroxidase: Depolymerization was carried out, as described above for Mr?‘, with 500 U/l manganese peroxidase and 0.5 mM MnSO, dissolved in 0.5 M sodium lactate, pH 4.5. H202 was continuously added at a rate of 7 pmol/min.l. Controls were run omitting either manganese peroxidase, Mn2+ or H202, or using 0.5 mM Mn3+ instead of Mn’+, manganese peroxidase, and H202. Manganese peroxidase was isolated from liquid cultures of P. chrysosporium lacking lignin peroxidase activity and purified by FPLC on a Mono Q column (Pharmacia-LKB). Analvsis of degradation oroducts: Size exclusion chromatography of chlorolignin was performed by HPLC using a Bio-Bad model 400 chromatograph and a TSK G3OOOSW column (7.5 x 600 mm, Pharmacia-LKB) with 0.03 M sodium acetate buffer, pH 6.7 and 0.5 g/l polyethylene glycol as the mobile phase at a flow rate of 1 ml/min. Bleach plant effluent (El), fractionated by ultra-filtration as well as blue dextrane and 4-chlorophenol were used to calibrate the column (Fig 2A). The color of the effluent was measured spectrophotometrically at 465 nm according to Eaton et al. (19) and expressed as color units (CU/l). The chemical oxygen demand (COD) was measured using a commercial assay kit (Dr. Lange, Austria) according to the supplier. 1093
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For weight loss determination the degraded HMW chlorolignin was dialyzed against water, concentrated by evaporation and dried at 60°C as well as at 110°C. Electronhoresis and immunoblottinp: The culture filtrates were concentrated 25 to 75-fold prior to electrophoresis (Amicon Centricon 10) and protein concentrations were measured using Bio-Rad’s Coomassie blue microassay. Polyacrylamide gel electrophoretic analysis (20) and immunoblotting (Western blotting) of ligninase proteins (lignin peroxidase and manganese peroxidase) were performed as described elsewhere (21). Enzvme assavs: Lignin peroxidase activity in culture fluids was measured spectrophotometrically using veratrylalkohol as a substrate (18) and manganese peroxidase was assayed with 2,bdimethoxyphenol according to Paszczynski et al. (3). One unit (II) of activity was defined as the oxidation of 1 pmol substrate per minute at 25°C for both enzymes. Manganese peroxidase was extracted with 0.5 M NaCl after washing of the immobilized fungus several times with destilled water. RESULTS
AND DISCUSSION
Fig. 1A shows lignin peroxidase and manganese peroxidase production, and degradation of high molecular weight chlorolignin in a semicontinuous culture of Phunerochaete chlysosporium. From day 5 to day 25, the culture fluid was decanted and replaced with fresh medium every day. The data, therefore, represent what was produced or degraded within 24 hours. Manganese peroxidase was measured directly in the culture fluid containing chlorolignin, whereas lignin peroxidase, for known methodical reasons, could be measured only in parallel cultures without chlorolignin. Interestingly, color decrease was still high after 25 days of cultivation (35%) although ligninolytic activity decreased to almost zero after 17 days. To ensure the absence of ligninase protein (lignin peroxidase and manganese peroxidase), the culture filtrate was concentrated 75-fold and separated by electrophoresis. No protein could be detected in 21 day old cultures (Fig. 1B) and immunoblotting did not show significant amounts of ligninase proteins (Fig. 1C). However, considerable amounts of manganese peroxidase (corresponding to
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(+) Ligninperoxidase production and (s) manganese production (U/l) and (*) of chlorolignin (910colour decrease)in semicontinuous liquid cultures of P. chysosporium [A]. Elecrophoretic separation [B] and immunoblotting [C] of the culture fluids after 5 days (lanes a, concentrated 25-fold) and 21 days (lanes b, concentrated75-fold) of cultivation. Lanes c: ligninase standard (control). decolonzahon
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50-100 U/l) but no lignin peroxidase was readily extractable from the mycelium with 0.5 M NaCl. This is in agreement with the finding of Paszczynski et al. (3) that manganese peroxidase is at least partly associated closely with the mycelium. Therefore, it seemed likely that mycelium bound manganese peroxidase might play an important role in bleach plant effluent degradation. To show this, we performed subsequent experiments during which the high molecular weight chlorolignin was not added directly to the cultures but packed into dialysis tubings and incubated with the fungus for several days. Decolorization of the effluent was observed (Fig. 3A) and the chlorolignin was depolymerized (Fig. 2B) although the fungal enzymes could not directly react
I3
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Eig-2. Gel chromatography of high molecularweightchloroligninin dialysistuhingsbefore (-) and after (---) treatmentwith P. chtysosporizun for 3 days[B], Mn3+for 2 days [Cl, and manganese peroxidasefor 3 days[D]. GraphA: calibrationof thecolumnwith bluedextran(l), 4-chlorophenol(5) andfractionatedbleachplant effluentwith m, > 30,000(2), 1000-5000(3), and < 1000(4).
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with the effluent due to the dialysis membrane. No manganese peroxidase could be detected inside the tubing whereas high activities were measured in the culture fluid outside the tubing. Due to diffusion limitation, a slower degradation rate than without dialysis tubing was observed. These results indicated that a low molecular weight agent degraded the polymeric bleach plant effluent. The small, diffusible manganese-(III)-complex, generated by manganese peroxidase, could be this reactive molecule. Using Mn3+-lactate as a biomimetic model for enzymatically generated Mn3+ (13), the oxidation of polymeric chlorolignin was also performed in dialysis tubings and a decolorization of about 80% was reached after 4-5 days of incubation (Fig. 3A). The manganese solution outside the tubing was changed every 12-15 hours because of the reported half life time of about 10 hours for Mn3+ due to hydrolyzation and disproportionation to Mn4’ (MnO*) and Mn’+ (13). After dialysis against water to remove interfering lactate and low molecular weight degradation products, a significant decrease in COD (60%) and dry weight (40%) could be measured (Table 1). The gel permeation elution profile was altered significantly and the adsorption at 280 nm (Fig. 2C) as well as at 465nm (not shown) decreased. Color decrease due to oxidation of the chromophores was twice as high as weight loss because only depolymerization products with molecular weights below 10,000-15,000 were removed by dialysis. In subsequent experiments, manganese peroxidase was used for Mn3’ generation from Mn2+ under optimum conditions for the enzyme (2). As the kinetics of chlorolignin oxidation by Mn3+ is not known, the rate of H202 addition had to be adjusted and 7 pmol/l.min at 37°C was found to be the optimum. The H,Oz was completely consumed and enzyme inactivation was low (15%/day) under these conditions. Degradation occured through the dialysis membrane indicating that a direct contact between enzymes and substrate (chlorolignin) was not necessary. Decolorization (Fig. 3A), weight loss (Table 1) and gel permeation profile (Fig. 2D) were similar to the enzyme-free Mn’+ -system, but COD decrease was lower, probably due to the
9. COLOR
DECREASE
100,
ORGAN,;
0
1
2 DAYS
3 OF INCUBATION
4
5
W,D
-.-
.--.-.
_.-_._.-A
6
Fig. 3. Comparison of chlorolignin decolorization in time by P. chrysosporium in vivo,
manganese peroxidase, and Mn3+ [A]. Proposed mechanism of chlorolignin depolymerization in the dialysis tubing system [B]. 1096
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Table l.Decolorization, COD decrease, and weight loss of high molecular weight chlorolignin in dialysis tubings after treatment with Mn3+ and manganese peroxidase in time DAYS
Mn-(III)-COMPLEX 0 1 2 3 5 MANGANESE 0 1 2 3 4 5
COLOR m/1
%
COD w/l
%
WEIGHT w/l
%
563 478 388 304 123
0 15 31 46 78
79 61 38 35 31
0 23 51 55 61
326 300 280 244 186
0 8 14 25 43
PREOXIDASE 626 538 400 238 194 175
0 14 36 62 69 72
53 46 44 39 36 34
0 12 17 26 31 36
373 347 328 298 265 231
0 7 12 20 29 38
different manganese concentrations and, therefore, redox potentials in the two systems. No degradation was observed when manganese peroxidase, Mn2+, or H202 was omitted. Furthermore, controls with 0.5 mM Mn3+ did not show any significant decolorization, indicating that Mn 3+ had been continuously regenerated by the enzyme. The rate of degradation was much higher with Mn3+ and manganese peroxidase in vitro than with P. chrysosporium in tivo in all experiments, indicating that manganese peroxidase together with H202 generating enzymes is solely responsible for the initial breakdown of high molecular weight chlorolignin in bleach plant effluents by P. chrysosporium in vivo. The conclusions which may be drawn from the results are summarized in Fig. 3B. Mn3+, generated by extracellular manganese peroxidase of the fungus, freely diffuses into the dialysis tubing to react with chlorolignin. Low molecular weight degradation products can then diffuse out of the tubing to be metabolized by the fungus and Mn3+ is regenerated from Mn*+. Recently, Hammel and Moer (8) have demonstrated the depolymerization of dehydropolymerizate (DHP) by lignin peroxidase and Wariishi et al. (9) have very recently published a paper showing that manganese peroxidase can also depolymerize DHP. However, lignin peroxidase was not necessary for depolymerization of the chlorolignins in our experiments. Although the chemical properties of chlorolignin are very different from DHP and native lignin, our results confirmed the significance of manganese peroxidase in the initial breakdown of polymeric substrates. This work was supported by the “Forschungsforderungsfonds gewerblichen Wirtschaft” . Acknowledgment:
REFERENCES
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