Protease inhibitors have great demand in medicine and biotechnology. We report here the purification and characterization of a protease inhibitor isolated from mature leaf extract of Moringa oleifera that showed maximum inhibitor activity. The protease inhibitor was purified to 41.4-fold by Sephadex G75 and its molecular mass was calculated as 23,600 Da. Inhibitory activity was confirmed by dot-blot and reverse zymogram analyses. Glycine, glutamic acid, alanine, proline and aspartic acid were found as the major amino acids of the inhibitor protein. Maximal activity was recorded at pH 7 and at 40 degrees C. The inhibitor was stable over pH 5-10; and at 50 degrees C for 2 h. Thermostability was promoted by CaCl(2), BSA and sucrose. Addition of Zn(2+) and Mg(2+), SDS, dithiothreitol and beta-mercaptoethanol enhanced inhibitory activity, while DMSO and H(2)O(2) affected inhibitory activity. Modification of amino acids at the catalytic site by PMSF and DEPC led to an enhancement in the inhibitory activity. Stoichiometry of trypsin-protease inhibitor interaction was 1:1.5 and 0.6 nM of inhibitor effected 50% inhibition. The low k(i) value (1.5 nM) obtained indicated scope for utilization of M. oliefera protease inhibitor against serine proteases. (C) 2011 Elsevier Ltd. All rights reserved.