Bijina, B; Sreeja, Chellappan; Soorej, Basheer M; Elyas, K K; Ali, Bahkali H(Elsevier, 2011)
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Abstract:
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 ◦C. The
inhibitor was stable over pH 5–10; and at 50 ◦C for 2 h. Thermostability was promoted by CaCl2, BSA
and sucrose. Addition of Zn2+ and Mg2+, SDS, dithiothreitol and -mercaptoethanol enhanced inhibitory
activity, while DMSO and H2O2 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 Ki value (1.5 nM)
obtained indicated scope for utilization of M. oliefera protease inhibitor against serine proteases