Protease inhibitor from Moringa oleifera leaves: Isolation, purification, and characterization

Abstract

a b s t r a c tProtease 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 CaCl 2 , BSA and sucrose. Addition of Zn 2+ and Mg 2+ , SDS, dithiothreitol and ␤-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.

Protease Inhibitor From White Cranberry Beans (Phaseolus Vulgaris): Isolation, Purification and Characterization

“…But sucrose at 70 °C did not support the thermal stability of inhibitor resulting thermal inactivation. Parallel results were obtained for Moringa leaf inhibitor [28]. Amino acids, salts and polyols have been reported to enhance thermal stability of proteins [29].…”

Purification, characterization, and bioassay of putative protease inhibitors from Hevea brasiliensis latex

“…One protease unit was defined as the amount of enzyme that increased absorbance by 1 OD/min and one protease inhibitor unit is defined as the amount of protease inhibitor that inhibited one unit of protease activity. The protease inhibitor activity was expressed in terms of percent inhibition (Bijina et al, 2011). It was calculated using the following formula used by Musor et al (2010): Percent inhibition (%) = {[Abs protease enzyme -(Abs sample -Abs sample blank)]/Abs protease enzyme} x 100%.…”

“…The increased use in agriculture sector as an antimicrobial agent has positioned plant protease inhibitors as an important product (Bijina et al, 2011;Islamov et al, 2012;Sack et al, 2015). Isolation and characterization of protease inhibitors from various plant species have been carried out using affinity column chromatography (Bijina et al, 2011;Popovic et al, 2013;Al-Assadi, 2013, Bunyatang et al, 2013Chaudhary et al, 2008;Sabotič et al, 2012).…”

“…Isolation and characterization of protease inhibitors from various plant species have been carried out using affinity column chromatography (Bijina et al, 2011;Popovic et al, 2013;Al-Assadi, 2013, Bunyatang et al, 2013Chaudhary et al, 2008;Sabotič et al, 2012). In H. brasiliensis, several types of protease inhibitors have also been successfully extracted from latex (Bangrak & Chotigeat, 2011;Kanokwiroon et al, 2008;Sritanyarat et al, 2006).…”

Estimation of Uncertainty of Trypsin Inhibitor Activity Measurement in Legume Crops

“…The presence of anti-nutritional factors, such as protease inhibitors, could decrease nutritional value of legume crops and limit their utilization in human and animal nutrition, but could also be beneficial to human health by preventing diseases such as cancer [4], [5], [6]. Depending on time and dose of consumption, protease inhibitor will have beneficial or anti nutritional effects [7].…”