Crude extracts were partially purified by organic solvents fractionation: best results (96% of proteins, 91% of total casemolytic activity) were obtained by adding four volumes of cold acetone to the crude extract. This preparation (redissolved acetone precipitate, RAP) showed maximum activity (> 80%) at pH 5-9, and exhibited high thermal stability (> 90% of residual activity after heating for 60 min at 60 degrees C). The enzyme was completely inhibited by E-64 trans-epoxy succinyl-leucyl -amido(4-guanidino)-butane and iodoacetic acid and activated by the addition of cysteine or beta-mercaptoethanol; these results strongly suggest that the isolated protease should be included within the cysteine group, as all the other studied proteases belonging to the family Bromeliaceae. IEF-zymogram of RAP showed five bands (pl 7.3 to < 9.3), most of them proteolytically actives, but only three of which (pI 7.6, 8.2 and 8.8) proved to be important. Ion exchange chromatography in DEAE-Sephadex, was selected to separate this active bands. (c) 2006 Elsevier Inc. All rights reserved.