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 MP activity was obtained in peptone medium, up to nearly 3 U/ml in cultures of P. eryngii. Moreover, Mn²⁺ supplementation was not used to produce MP, since all Mn²⁺ concentrations assayed (1–4000 μM) inhibited production of this enzyme in liquid medium.Two MP isoenzymes were purified to homogeneity from shaken or stationary cultures of P. 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 Mn²⁺ ‐independent peroxidase activities against 2,6‐dime‐thoxyphenol and veratryl alcohol, and higher resistance to H₂O₂. Recent studies have shown that MP 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 pI 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_m= 0.84 mM, V_max= 52 U/mg) to 4‐methoxybenzyl alcohol (K_m= 0.04 mM, V_max= 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.

A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe ³⁺ -EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H ₂ O ₂ + Fe ²⁺ → OH + OH ⁻ + Fe ³⁺ ). H ₂ O ₂ production, Fe ³⁺ reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione [MD]). In all cases, 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 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 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 OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.