Purification and Characterization of a Pineapple Crown Leaf Thiol Protease

Abstract

A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico‐chemical and kinetic properties. The molecular weight of the protease (22.4–22.9 kDa), K m (97 µM) and k cat (8.8 s−1) for its esterolytic cleavage of the synthetic protease substrate N α‐CBZ‐L‐lysine p‐nitrophenyl ester, the concentration of its thiol activator L‐cysteine required for half maximal activation A 0.5 (9.9 µM), optimum pH (6.5) for its proteolytic action on azocasein, T 1/2 (60°C) for inactivation by heating the enzyme (35.5 µg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH‐group content (0.98 mol/mol enzyme) were determined. Most of these physico‐chemical and kinetic properties were found to be similar to those of the already well‐characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.