Purification and characterization of macrodontain I, a cysteine peptidase from unripe fruits of Pseudananas macrodontes (Morr.) Harms (Bromeliaceae)

A new papain-like cysteine peptidase isolated from fruits of Pseudananas ma crodontes (Morr.) Harms, a species closely related to pineapple (Ananas com osus L.), has been purified and characterized. The enzyme, named macrodonta in I, is the main proteolytic component present in fruit extracts and was p urified by acetone fractionation followed by anion-exchange chromatography. Separation was improved by selecting both an adequate pH value and a narro w saline gradient. Optimum pH range (more than 90% of maximum activity with casein) was achieved at pH 6.1-8.5. Homogeneity of the enzyme was confirme d by bidimensional electrophoresis and mass spectroscopy (MS). Molecular ma ss of the enzyme was 23,459 (MS) and its isoelectric point was 6.1. The ala nine, glutamine, and tyrosine derivatives were strongly preferred when the enzyme was assayed on N-alpha-CBZ-L-amino acid p-nitrophenyl esters. The N- terminal sequence of macrodontain (by comparison with the N-terminus of 30 plant proteases with more than 50% homology) showed a great deal of sequenc e similarity to the other pineapple-stem-derived cysteine endopeptidases, b eing 85.7, 85.2, and 77.8% identical to comosain, stem bromelain, and anana in, respectively. It seems clear that the Bromeliaceae endopeptidases are m ore closely related to each other than to other members of the papain famil y, suggesting relatively recent divergence. (C) 2000 Academic Press.