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.

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.

Spectral and catalytic properties of the flavoenzyme AAO (aryl-alcohol oxidase) from Pleurotus eryngii were investigated using recombinant enzyme. Unlike most flavoprotein oxidases, AAO does not thermodynamically stabilize a flavin semiquinone radical and forms no sulphite adduct. AAO catalyses the oxidative dehydrogenation of a wide range of unsaturated primary alcohols with hydrogen peroxide production. This differentiates the enzyme from VAO (vanillyl-alcohol oxidase), which is specific for phenolic compounds. Moreover, AAO is optimally active in the pH range of 5-6, whereas VAO has an optimum at pH 10. Kinetic studies showed that AAO is most active with p-anisyl alcohol and 2,4-hexadien-1-ol. AAO converts m- and p-chlorinated benzyl alcohols at a similar rate as it does benzyl alcohol, but introduction of a p-methoxy substituent in benzyl alcohol increases the reaction rate approx. 5-fold. AAO also exhibits low activity on aromatic aldehydes. 19F NMR analysis showed that fluorinated benzaldehydes are converted into the corresponding benzoic acids. Inhibition studies revealed that the AAO active site can bind a wide range of aromatic ligands, chavicol (4-allylphenol) and p-anisic (4-methoxybenzoic) acid being the best competitive inhibitors. Uncompetitive inhibition was observed with 4-methoxybenzylamine. The properties described above render AAO a unique oxidase. The possible mechanism of AAO binding and oxidation of substrates is discussed in the light of the results of the inhibition and kinetic studies.

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH 2 ). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH 2 extended the reactions catalyzed by these enzymes to the production of H 2 O 2 , the oxidation of Mn 2؉ , the reduction of Fe 3؉ , and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.Lignin is an aromatic heteropolymer of phenyl-propanoid units which confers structural rigidity to woody plant tissues and protects them from microbial attack (25). To depolymerize and mineralize lignin, white rot fungi have developed a nonspecific oxidative system including several extracellular oxidoreductases, low-molecular-weight metabolites, and activated oxygen species (47). The ability of white rot fungi to degrade a wide number of organopollutants is in part due to the action of this nonspecific system (42). Extracellular enzymes involved in the degradation of lignin and xenobiotics by white rot fungi include several kinds of laccases (34, 49), peroxidases (8, 33), and oxidases producing H 2 O 2 (20,32,50).The enzymatic composition of the ligninolytic system depends on the fungal species, with laccase being the common component (24,43). For this reason, a wide number of studies have focused on demonstrating the participation of laccase in significant ligninolytic events which were first attributed to other enzymes of the ligninolytic system. These events include the oxidation of nonphenolic lignin units, which comprise ca. 80% of the polymer, the generation of the H 2 O 2 required for both peroxidase activities and hydroxyl radical ( ⅐ OH) formation, and the production of Mn 3ϩ from the Mn 2ϩ pr...

. In all cases, ⅐ OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H 2 O 2 levels and Fe 3؉ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher ⅐ OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between ⅐ OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of ⅐ OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.

Two protein bands with laccase activity were found after PAGE of culture liquid or mycelium extract of Pleurotus eryngii, grown on glucose-ammonium tartrate-yeast extract medium with and without inducers. A major and a minor laccase band were observed in the basal medium. The intensity of the major band (laccase I) did not change after the addition of inducers. However, the minor band (laccase II), characterized by higher electrophoretic mobility, was strongly induced by wheat-straw alkalilignin and vanillic and veratric acids. Laccase activity in the basal medium had an optimum pH of 4.5 and was stable from pH 3 to 10 during 24 h at room temperature. This enzyme had wide substrate specificity on hydroquinones, methoxy-substituted monophenols, and aromatic amines. In general, laccase activity was found only with compounds having a redox potential lower than 0.5 mV. The highest activity was obtained with methoxy- and methyl-substituted p-hydroquinones and aromatic diamines. Some activity also occurred with the aliphatic compound 3,5-cyclohexadiene-1,2-diol.

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Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH 2 ) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH 2 ) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH 2 more efficiently than it oxidized MBQH 2 ; both the affinity and maximal velocity of oxidation were higher for DBQH 2 than for MBQH 2 . Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (