Kinetic differences of puri®ed laccases from six Pleurotus ostreatus strains R.Tinoco 1 , M.A. Pickard 2 and R. Vazquez-Duhalt 1 1 Instituto de Biotecnologia UNAM, Morelos. Mexico,and 2 Department of Biological Sciences, University of Alberta, Edmonton, Canada 2000/56: received 8 February 2001 and accepted 16 February 2001 R. TINOCO, M.A. PICKARD AND R. VAZQUEZ-DUHALT. 2001. Aims: Enzyme kinetics of puri®ed laccases from six different Pleurotus ostreatus strains were determined in the oxidation of syringaldazine, guaiacol and ABTS. Methods and Results: Signi®cant differences in the kinetic constants were found. Catalytic activity (k cat ) ranged from 19 to 941 U mg )1 for syringaldazine, from 18 to 1565 U mg )1 for ABTS, and from 4 to 44 U mg )1 for guaiacol. The apparent af®nity constants (K M ) also showed signi®cant differences between the different strains, from 12 to 52 lmol l )1 for syringaldazine, from 8 to 79 lmol l )1 for ABTS, and from 0á46 to 6á61 mmol l )1 for guaiacol. No differences were found either on the effect of increasing concentrations of organic solvent (acetonitrile) or on the activity pH pro®le. The temperature pro®le was the same for all the P. ostreatus strains, except for the IE8 strain, which seems to be more sensitive to temperature. The kinetic and stability data from the six P. ostreatus strains were also compared with those obtained from other white rot fungi, Coriolopsis gallica and Trametes versicolor, showing clear differences. Conclusions: The different P. ostreatus isolates showed different kinetic constants. Signi®cance and Impact of the Study: The different enzymatic properties of laccases from various P. ostreatus strains should be considered for a potential industrial or environmental application. INTRODUCTION Laccases (EC 1.10.3.2; benzenediol:oxygen oxidoreductases) from white rot fungi are part of the complex enzymatic system for lignin degradation (Hatakka 1994). Laccases catalyse the one-electron oxidation of a wide range of phenolic and aromatic amines (Thurston 1994). The use of laccase- mediator systems are promising alternatives for environ- mental biotechnological processes such as pulp-bleaching (Bourbonnais et al. 1995; Li et al. 1998), textile dye decolori- zation (Reyes et al. 1999; Rodriguez et al. 1999) and oxidation of polycyclic aromatic hydrocarbons (Bohmer et al. 1998; Pickard et al. 1999a). Laccases are blue multi-copper oxidases found in plants and fungi. Laccases contain three Cu-centres; T1, T2 and T3 sites. The T2 and T3 sites form a trinuclear Cu cluster at which O 2 is reduced. The T1 Cu-centre oxidizes the reducing substrate and transfers the generated electrons to the T2 and T3 Cu-centres (Farver and Pecht 1984; Messerschmidt and Huber 1990). The catalysis involves the binding of a reducing substrate to the T1 pocket and subsequent reduction of the Cu(II) to Cu(I) in the T1 site, then internal electron transfer from T1 to the T2/T3 cluster, and ®nally, the binding and subsequent reduction of an O 2 to H 2 O at the T2/T3 cluster (Xu 1997; McGuirl and Dooley 1999). Laccases from different fungi show signi®cant variations in their properties, such as redox potentials, kinetic constants, substrate speci®city, optimal pH (Xu 1997; Heinzkill et al. 1998; Xu et al. 2000). In addition, site-directed mutations of speci®c residues can greatly affect the redox potential, activity and pH pro®le (Xu et al. 1998). The white rot fungus Pleurotus ostreatus is able to metabolize a variety of pollutant xenobiotics, such as polychlorinated biphenyls (PCB) (Beau- dette et al. 1998), polycyclic aromatic hydrocarbons (PAHs) Correspondence to: Dr Rafael Vazquez-Duhalt, Instituto de Biotecnologõ Âa UNAM, Apartado Postal 510±3, Cuernavaca, Morelos, 62250 Mexico (e-mail: vazqduh@ibt.unam.mx). ã 2001 The Society for Applied Microbiology Letters in Applied Microbiology 2001, 32, 331±335