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Purification And Characterization Of Papain Inhibitors From Amaranto (Amaranthus Caudatus) And Frijol (Phaseolus Vulgaris) Seeds

Abstract:

Plant protease inhibitors are often low molar mass proteins and are present in plant storage and aerial tissues. Their expression is induced in response to damage by insects and pathogenic microorganisms, and the content of these inhibitors can be high in cereal and legume organs. Under the premise that the combination of low and high resolution protein purification techniques allows increasing the specific activity of protease inhibitors present in plant tissues, the objective of this study was to isolate, purify, and biochemically characterize papain inhibitors from amaranth (Amaranthus caudatus) and bean (Phaseolus vulgaris) seeds. Completely randomized experimental designs were used to select low resolution techniques that significantly increase (p£0.05) the purification degree of the treated extracts. In all studies, three replicates were made, and measurements were performed in triplicate. Crude protein extracts were obtained from amaranth and bean seed flours, which were then ultrafiltered in 10 kDa membranes. Low molar mass fractions were purified by affinity chromatography in papain-glyoxyl-sepharose 6B-CL. The crude amaranth extract presented inhibitory activity of 1.177 ± 0.067 mU mL-1, and the bean extract presented an inhibitory activity of 1.556 ± 00.542 mU·mL-1. The amaranth and bean extracts with partial purification were purified by affinity chromatography in papain-glioxyl-sepharose 6B-CL, up to 137.5 and 79.1 times, respectively. The estimated molar mass was 7.50 kDa for the amaranth inhibitor, and 8.20 kDa for the bean inhibitor. The amaranth inhibitor showed competitive inhibition with 0.872 mM Ki, while the bean inhibitor was non-competitive with 0.058 mM Ki. This demonstrates the presence of cysteine protease inhibitors in amaranth and bean seeds. masa molar estimada fue 7.50 kDa para el inhibidor de amaranto y 8.20 kDa para el de frijol. El inhibidor de amaranto mostró inhibición competitiva con Ki de 0.872 mM, mientras que la del frijol fue no competitiva y con Ki de 0.058 mM. Esto evidencia la presencia de inhibidores de cisteíno proteasas en las semillas de amaranto y frijol.