Purification and Characterization of a Novel Thermostable Papain Inhibitor from Moringa oleifera with Antimicrobial and Anticoagulant Properties

Abstract

Plant cystatins (or phytocystatins) comprise a large superfamily of natural bioactive small proteins that typically act as protein inhibitors of papain-like cysteine proteases. In this report, we present the purification and characterization of the first phytocystatin isolated from Moringa oleifera (MoPI). MoPI has a molecular mass of 19 kDa and showed an extraordinary physicochemical stability against acidic pHs and high temperatures. Our findings also revealed that MoPI is one of the most potent cysteine protease inhibitors reported to date, with Ki and IC₅₀ values of 2.1 nM and 5.7 nM, respectively. More interestingly, MoPI presents a strong antimicrobial activity against human pathogens such as Enterococcus faecalis and Staphylococcus aureus. In addition, MoPI also showed important anticoagulant activity, which is an unprecedented property for this family of protease inhibitors. These results highlight the pharmaceutical potential of this plant and its derived bioactive molecules.

Keywords: Moringa oleifera; antibacterial; anticoagulant; bioactive compound; papain inhibitor; phytocystatin; plant protease inhibitor; protease.

Figure 1

Isolation and purification of M. oleifera papain inhibitor (MoPI) from the crude extract (MoCE). (A) Papain inhibitory activity (%) in the presence of the different M. oleifera heat-treated samples. MoCE: M. oleifera crude extract, MoHT15-120: M. oleifera crude extract after 100 °C heat treatment for 15, 60 and 120 min. * p < 0.05, compared to control; ^a p < 0.05 compared to MoCE; ^b p < 0.05 compared to MoHT15; ^c p < 0.05 compared to MoHT60 (one-way ANOVA and Tukey´s multiple comparison test). (B) Purification of M. oleifera papain inhibitor by affinity chromatography on immobilized papain. Fractions with papain inhibitory activity (gray area) were pooled and named MoPI. (C) Electrophoresis (SDS-PAGE, 12% v/v) of the M. oleifera papain inhibitor (MoPI). Lane 1: Low range molecular mass marker; Lane 2: Purified M. oleifera papain inhibitor. The arrow indicates the location of the inhibitor.

Figure 2

Kinetic studies on M. oleifera papain inhibitor. (A) Dose response curve for the half maximal inhibitory concentration (IC₅₀) determination. (B) Dixon plot (1/v vs. [I]) for identification of the Ki. Each point represents the mean of three estimates.