A Proteinaceous Alpha-Amylase Inhibitor from Moringa Oleifera Leaf Extract: Purification, Characterization, and Insecticide Effects against C. maculates Insect Larvae

Abstract

The main objective of the current study was the extraction, purification, and enzymatic characterization of a potent proteinaceous amylase inhibitor from Moringa oleifera. The antimicrobial potential and insecticide effects against C. maculates insect larvae were also studied. The α-amylase inhibitor was extracted in methanol (with an inhibitory activity of 65.6% ± 4.93). Afterwards, the inhibitor αAI.Mol was purified after a heat treatment at 70 °C for 15 min followed by one chromatographic step of Sephadex G-50. An apparent molecular weight of 25 kDa was analyzed, and the N-terminal sequence showed the highest identity level (89%) with the monomeric α-amylase inhibitor from Triticum dicoccoides. αAI.Mol was found to tolerate pH values ranging from 5.0 to 11.0 and showed maximal activity at pH 9.0. Thermal stability was remarkably important, since the inhibitory activity was maintained at 55% after 1 h of incubation at 70 °C and at 53% after an incubation of 45 min at 80 °C. The potency of the current purified inhibitor against amylases from different origins indicates that αAI.Mol seems to possess the highest affinity toward human salivary α-amylase (90% inhibitory activity), followed by the α-amylase of insects Callosobruchus maculatus and Tribolium confusum (71% and 61%, respectively). The kinetic parameters were also calculated, and the Kmax and Vmax of the digestive amylase were estimated at 185 (mmol/min/mg) and 0.13 mM, respectively. The inhibitor possesses a strong bactericidal effect against Gram+ and Gram- strains, and the MIC values were >1 against B. cereus but >6 against E. coli. Interestingly, the rates of survival and pupation of C. maculates insect larvae were remarkably affected by the purified αAI.Mol from Moringa oleifera.

Keywords: insecticide effects; kinetic parameters; plant defense; α-amylase inhibitor.

Figure 1

α-Amylase inhibitor activity extracted from Moringa oleifera using different solvents.

Figure 2

Purification of αAI.Mol: (A) gel filtration chromatography on Sephadex G-50. Column of G-50 (2.5 × 100 cm) equilibrated with 0.1 Μ Tris-HCL buffer, pH 8, containing 0.2 Μ NaCl. The column was eluted with the same buffer at a flow rate of 30 mL/h, and 4.55 mL fractions were collected. Red line: active fractions. (B) SDS-PAGE analysis. Twenty micrograms of the active fractions containing the amylase inhibitor activity.

Figure 3

(A,B) Effect of pH and temperature on αAI.Mol activity: Effect of pH on PDInhibitor activity (A) and stability (B). Inhibitor activity was assayed at various pH levels, and inhibitor stability was tested after an incubation of the αAI.Mol at different pH levels for 12 h. (C,D) Effect of temperature on αAI.Mol activity (C) and stability (D). Inhibitor activity was assayed at various temperatures (50 °C–90 °C). For stability studies, the amylase inhibitor was incubated at different temperatures and drawn at various time intervals and assayed for residual inhibitor activity at optimal conditions of pH and temperature. Data shown are mean ± SD (n = 3).

Figure 4

Effects of metal ions at concentrations of 1 and 5 mM on αAI.Mol activity. The α-amylase inhibitor assay was performed at 45 °C and pH 8. The control represents 100% of the α-amylase inhibitor activity under the same condition in the absence of any metal. Data shown are mean ± SD (n = 3).

Figure 5

Antibacterial properties of the alpha amylase inhibitor (αAI.Mol) from Moringa oleifera). The antibacterial effect was evaluated against several Gram-positive and Gram-negative bacteria presented by zone of inhibition (mm) using pure amylase inhibitor (gray bars) and crude extract (black bars).

Figure 6

Bioassays of purified amylase inhibitor (αAI.Mol) from Moringa oleifera against C. maculates insect larvae: pupation and mortality. (A) Rate of pupation of C. maculates insect larvae. The bioassay was studied by feeding C. maculatus larvae a diet containing purified αAI.Mol and compared to a control of C. maculatus larvae on a diet without the inhibitor αAI.Mol (gray bars). Pupation was recorded every 24 h and continued for up to 5 days. The experiment was repeated in triplicate, and standard deviations were recorded. (B) Rate of mortality of C. maculates insect larvae. Mortality of larvae fed the αAI.Mol-containing diet as compared with the control of C. maculatus larvae on a diet without the inhibitor αAI.Mol (gray bars) diet was studied for up to 5 days.

Figure 7

Lineweaver Burk curves: the hydrolysis rates (V) of pure starch (S) at various concentrations [0–1.5 mM] at 45 °C, pH 8, for 15 min using α-amylase incubated with 10 µg of inhibitor (+αAI.Mol: green square) or without the inhibitor (−αAI.Mol: black square).