World Journal of Microbiology & Biotechnology 11, 497501 Sensitivity and specificity of phenoloxidase reactions of 1059 strains and species of Micromycetes cultivated on malt/agar medium M. Rahouti, J.-L. Benoit-Guyod, F. Seigle-Murandi* and P. Guiraud The phenoloxidase (POX) activities of 1059 strains and species of micromycetes were determined on malt/agar medium. Overall, 600 (5 7%) of the isolates produced one or more POX. The sensitivity and specificity of the POX activities towards various substrates were used to group the isolates. Some 187 strains (31% of those producing POX) produced well-defined enzymes, 236 (39%) produced incompletely identified enzymes and 177 (30%) produced other, unidentified POX. Key words: Fungi imperfecti, laccases, Micromycetes, peroxidases, phenoloxidases, tyrosinases. Phenoloxidases (POX), which are widely distributed in plants and microorganisms (Bollag & Leonowicz 1984, comprise three types of enzyme: laccases; peroxidases; and tyrosinases. As POX play a major role in the degradation of lignin and its phenolic components, most have received considerable attention (Eriksson et al. 1990). Research on peroxidases developed after ligninase was discovered by three research groups (Glenn et al. 1983; Shimada & Higuchi 1983; Tien & Kirk 1983) but tyrosinases have received relatively little attention (Marr et al. 1986; Ingebrigtsen et al. 1989). The metabolic activity of Micromycetes towards various phenolic compounds (fen&c acid, vanillic acid and pentachlo- rophenol) and their ability to produce POX vary with the strain studied (Rahouti et al. 1989; Guiraud et al. 1992; Seigle-Murandi et al. 1992). It would be interesting to determine which type of POX each strain produces and how this relates to metabolic activity. In the present study, 1059 strains of Micromycete were M. Rahouti is with the Labor&ok de Microbiologic, Facult& des Sci- ences, Avenue Ibn Batouta. BP 1014, Rabat, Morocco. J.-L. Benoit-Guyed, F. Seigle-Murandi and P. Guiraud are with the Groupe pour I’ctude du Devenir des XBnobiotiques dans I’Environnement (GEDEXE), UFR de Phar- macie de Grenoble, Universitb Joseph Fourier, BP 138, 36243 Meylan. France; fax: 33 76 04 10 05. ‘Corresponding author. cultivated on malt/agar medium and their activities towards various phenols or aromatic amines were determined. Sub- strate specificities and sensitivities were compared between strains and an attempt was made to identify the POX produced by each strain. Materials and Methods Microorganisms All the fungal strains used (see Table 1) were from the mycology collection of the Pharmacie de Grenoble, Meylan, France. Most are ‘fungi imperfecti’ isolated from various substrates, including soil, decayed wood and food. Phenoloxidase Detection Each strain was grown on 1.5% (w/v) malt extract/agar for 12 days at 24°C. POX were detected by direct application of reagents to the mycelium (Guiraud et al. 1992; Seigle-Murandi et al. 1992). Solutions (0.1 M in 95% ethanol) of the reagents [benzidine (Ben), guaiacol (Gua), o-anisidine (o-An), pyrogallol (Pyr), a-naphthol (a- Na), p-cresol @.Cr), tyrosine (Tyr) and gallic acid (GaA)] were as described by KZrik (1965). Syringaldazine (Syr) was used as a 0.1% (w/v) solution in 95% ethanol to which 0.03% H,O, in water could be added to demonstrate peroxidase activity (Harkin & Obst 1973). The R56 reagent used was 11.5% amidopyrine, 2.0% N-N diethylaniline and 2.5% benzoic acid in 95% ethanol (Dagron 1985). Reactions were read after 20 min (Syr) or 24 h (all other reagents). Drops of 95% ethanol applied to some mycelia World Journal of Mic&iology & Biotechnology. Vol I I, 1995 497