DECOLORIZATION OF AZO DYE CROCEIN ORANGE G BY PHANEROCHAETE CHRYSOSPORIUM LIGNIN PEROXIDASES.

Maarit Heikkilä¹, Pauli Ollikka² and Ilari Suominen^1,3, ¹Deparment of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, FIN-40351 Jyväskylä, Finland; ²Department of medical biochemistry, University of Turku, Kiinanmyllynkatu 10, FIN-20520 Turku; ³Department of Biochemistry, University of Turku, Vatselankatu 2, FIN-20014 Turku, Finland

A wide variety of dyes are used by industry and released into the environment in industrial effluents. These dyes have to be highly stable in everyday use and resistant to microbial degradation. Azo dyes are the largest class of dyes used in industry. In general, bacteria are not able to degrade azo dyes. However, some anaerobic bacteria in intestinal microflora have been demonstrated to degrade a few azo dyes. Under these conditions the azo linkage is reduced to generate aromatic amines that are colorless but can also be toxic and potentially carcinogenic. White rot fungus Phanerochaete chrysosporium has been shown to be able to degrade many aromatic pollutants, including azo dyes, by it's ligninolytic system. Degradation has been investigated either by whole culture of the fungus or by purified peroxidases.

Catalytic cycle of lignin peroxidase is common to peroxidases. First H₂O₂ oxidases native lignin peroxidase to compound I with two electrons. Substrate is oxidased in two steps when compound I is reduced to compound II and compound II is reduced back to the native form. Lignin peroxidase can also exist in the form of compound III, which does not take part in the catalytic cycle. This form is inactive and can either inactivate irreversibly or reverse back to the native lignin peroxidase in different situations. Compound III is formed for example from compound II in the excess of H₂O₂.

In this study we have shown that lignin peroxidase isoenzymes are able to degrade certain azo dyes. All investigated azo dyes (Acid Red 88, Crocein Orange G, Chicago Sky Blue 6 B and Congo Red) were degraded to some extent. The ability to degrade azo dyes varied between lignin peroxidase isoenzymes and optimum pH for decolorization differed between dyes.

Kinetics and optimal conditions of degradation of azo dye Crocein orange G (COG) was investigated in more detail. According to our results, the optimal concentration to decolorize 100 µM COG was 150 µM H₂O₂. This was true regardless of the amount of lignin peroxidase used in the reaction. Oxidation of COG by lignin peroxidase and H₂O₂ appeared to follow Michaelis-Menten kinetics. Our results also suggest that H₂O₂ and COG compete for lignin peroxidase. The mechanism with which H₂O₂ disturbed the reaction was supposed to be compound III formation by reaction of the compound II with H₂O₂. A similar observation has been previously made during the oxidation of phenols.

Previously it has been shown that during the oxidation lignin peroxidase is inactivated at low pH. In this study we have shown that when the concentration of H₂O₂ was higher than optimal, the inactivating effect of H₂O₂ exceeded the inactivating effect of pH. Because of this effect of H₂O₂ to lignin peroxidase, it is important to optimize the ratio of lignin peroxidase to H₂O₂. This is particularly important when biotechnological applications for decolorization of industrial effluents are planned.