Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae)

A new plant endopeptidase was obtained from unripe fruits of Bromelia balan sae Met (Bromeliaceae). Crude extracts were partially purified by ethanol f ractionation. This preparation (redissolved ethanol precipitate, REP) showe d maximum activity at pH 8.8-9.2, was very stable even at high ionic streng th values (no appreciable decrease in proteolytic activity could be detecte d after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibite d high thermal stability (inactivation required heating for 60 min at 75 de grees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balans ain I, with pi 5.45 and molecular mass 23192 (mass spectrometry). The purif ication factor is low (2.9-fold), but the yield is high (48.3%), a common o ccurrence in plant organs with high proteolytic activity, where proteases r epresent the bulk of protein content of crude extracts. Balansain I exhibit s a similar but narrower pH profile than that obtained for REP, with a maxi mum pH value similar to 9.0 and was inhibited by E-64 and other cysteine pe ptidases inhibitors but not affected by inhibitors of the other catalytic t ypes of peptidases. The alanine and glutamine derivatives of N-alpha-carbob enzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzy me. The N-terminal sequence of balansain I showed a very high homology (85- 90%) with other known Bromeliaceae endopeptidases.