Understanding the Substrate specificity of tyrosinases (EC 1.14.18.1) as well as their capability to oxidize peptide-bound tyrosine residues is important in a view of applicability of tyrosinases. In the present study, two fungal tyrosinases, an extracellular enzyme from the filamentous fungus Trichoderma reesei (TrT) and an intracellular enzyme from the edible mushroom Agaricus bisporus (AbT) were compared. Oxidation of various mono-and diphenolic compounds and tyrosine-containing tripeptides was examined and kinetic constants determined using spectrophotometric and oxygen consumption measurements. TrT and AbT were found to show notable differences in their substrate specificity. TrT generally showed 10-fold higher K values than AbT. The presence of a carboxylic and amine group in the substrate influenced the enzymes differently. While the substrates with a carboxyl group were observed not to be effectively oxidized by AbT, the amine group seemed to hider the oxidation in the TrT-catalyzed reactions. Moreover, the UV-visible absorption spectra on the oxidation of catechol and hydrocaffeic acid showed that the product patterns were different between the enzymes. The result is interesting as the primary products from tyrosinase-catalyzed reactions were assumed to be identical with both enzymes. Furthermore, a nucleophilic 3-methyl-2-benzothiazolinone hydrazone (MBTH) affected differently on the activity of the tyrosinases: the lag period related to the oxidation of monophenols was prolonged by MBTH with TrT, whereas with AbT the lag was shortened. (C) 2008 Elsevier Inc. All rights reserved.