Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH-compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (<50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.Laccases are multicopper oxidases that catalyze the oneelectron oxidation of substituted phenols, anilines, and aromatic thiols to their corresponding radicals with the concomitant reduction of molecular oxygen to water. Laccases are produced by plants and fungi, including white-rot basidiomycetes responsible for lignin degradation in nature (36, 46), although some bacterial laccases have been recently described and fully characterized (17). It is because of the involvement of laccases in lignin degradation that they were first investigated for applications in the pulp and paper industry as substitutes for chlorine-containing reagents for pulp bleaching (38, 41).The broad substrate specificities of laccases, together with the fact that they use molecular oxygen as the final electron acceptor instead of the hydrogen peroxide used by ligninolytic peroxidases (38), make these enzymes highly interesting for the pulp and paper industry as well as for other industrial and environmental applications. However, the low redox potentials of laccases (0.5 to 0.8 V) compared to those of ligninolytic peroxidases (Ͼ1 V) only allow the direct degradation by laccases of low-redox-potential phenolic compounds and not the oxidation of the most recalcitrant aromatics, including different industrial dyes (49). Nevertheless, insights into lignin d...

The ligninolytic basidiomycetes Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus sajor-caju did not exhibit detectable levels of manganese peroxidase (MP) when grown in liquid media with ammonium tartrate as N source. However, after examination of cells grown on different organic N-based media, high M P activity was obtained in peptone medium, up to nearly 3 U/ml in cultures of I? eryngii. Moreover, Mnz+ supplementation was not used to produce MP, since all Mn2+ concentrations assayed (1 -4000 pM) inhibited production of this enzyme in liquid medium.Two MP isoenzymes were purified to homogeneity from shaken or stationary cultures of I? eryngii grown in peptone medium. The purification process (which included chromatography on Biorad Q-cartridge, Sephacryl S-200 and Mono-Q) attained 56% activity yield with a purification factor of 25. The isoenzymes differed in PI (3.75 and 3.65), N-terminal sequence and some catalytic properties. They were in some aspects (e.g., molecular mass of 43 kDa) similar to Phanerochaete chrysosporium MP but exhibited some distinct characteristics, including Mn"-independent peroxidase activities against 2,6-dimethoxyphenol and veratryl alcohol, and higher resistance to H,O,. Recent studies have shown that M P are ubiquitous enzymes in ligninolytic fungi, but the results obtained suggest that differences in catalytic properties probably exist between different Mn2 ' -oxidizing peroxidases produced by these fungi.

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The production in a 5-1 fermenter of the extracellular enzymes laccase and aryl-alcohol oxidase by the fungus Pleurotus eryngii was studied. The latter enzyme has been purified 50-fold by Sephacryl S-200 and Mono Q chromatography. Purified aryl-alcohol oxidase is a unique flavoprotein with 15% carbohydrate content, a molecular mass of 72.6 kDa (SDS/PAGE) and a p l of 3.9. The enzyme presents wide specificity, showing activity on benzyl, cinnamyl, naphthyl and aliphatic unsaturated alcohols. Neither activity nor inhibition of veratryl alcohol oxidation was found with saturated alcohols, but competitive inhibition was produced by aromatic compounds which were not arylalcohol oxidase substrates, such as phenol or 3-phenyl-1-propanol. From these results, it was apparent that a double bond conjugated with a primary alcohol is necessary for substrate recognition by arylalcohol oxidase, and that activity is increased by the presence of additional conjugated double bonds and electron donor groups. Both affinity and maximal velocity during enzymic oxidation of methoxybenzyl alcohols were affected in a similar way by ring substituents, increasing from benzyl alcohol ( K , = 0.84 mM, V,,, = 52 Ujmg) to 4-methoxybenzyl alcohol ( K , = 0.04 mM, V,,, = 208 U/mg). Aryl-alcohol oxidase presents also a low oxidase activity with aromatic aldehydes, but the highest activity was found in the presence of electron-withdrawing groups.