Diseño de un sistema de purificación de inhibidores de papaína, provenientes de semillas de amaranto, arveja, chocho, fréjol, quinua o sangorache, mediante cromatografía de afinidad

Abstract

Papain inhibitors extracts from seeds were purified and characterized by affinity chromatography with papain-glyoxyl-sepharose 6B-CL. In the first phase, a completely randomized design was used to study the effect of pH of immobilization in four levels (8, 9, 10 and 11). A pH value of 8 was selected after analyzing the percent retention of functional activity (79.42%). Also, with a completely randomized design reduction treatments with NaBH4 were evaluated with the concentration and the addition form of the reductant. The best reduction process was with 5 mg NaBH4/mL in solution, which had a retention percentage of functional activity equal to 80.67%. The aqueous extracts of amaranth and bean showed higher specific inhibitory activity of papain with 0.279 and 0.259 mU/mg, respectively. A partial purification system for the crude amaranth extract was designed with ultrafiltration at 10 kDa and precipitation with ammonium sulfate. The partial purification system for the bean extract consisted of heat treatment at 60°C and centrifugal ultrafiltration at 10 kDa. Then, the fractions were purified by affinity chromatography on papain-glyoxyl-sepharose support. The kinetic and molecular characterization determined the presence of a competitive inhibitor of 7.20 kDa in the amaranth extract and a noncompetitive inhibitor of 8.50 kDa in the bean extract.