Biochemical characterization of purified ANCUT2 cutinase from Aspergillus nidulans is described. The identified amino acid sequence differs from that predicted in Aspergillus genomic databases in amino acids not relevant for catalysis. The enzyme is thermo-alkaline, showing its maximum activity at pH 9 and 60 degrees C, and it retains more than 60% of its initial activity after incubation for 1 h at 60 degrees C for pH values between 6 and 10. ANCUT2 is more active towards long-chain esters and it hydrolyzes cutin; however, it also hydrolyzes short-chain esters. Cutinase is inhibited by metal ions, PMSF, SDS, and EDTA (10 mM). It retains 50% of its activity in most of the solvents tested, although it is more stable in hydrophobic solvents. According to its found biochemical properties, preliminary assays demonstrate its ability to synthesize methyl esters from sesame oil and the most likely application of this enzyme remains in detergent formulations.