A heat‐stable trypsin inhibitor in adzuki bean (Vigna angularis): effect of extraction media, purification and biochemical characteristics

Summary Trypsin inhibitor from adzuki bean ( Vigna angularis ) seed was isolated and characterised. Extraction of seed with NaCl at the concentration of 0.15 m showed a higher recovery of trypsin inhibitor than other solvents tested ( P < 0.05). Optimal extraction time for the recovery trypsin inhibitor from adzuki bean seed was 30 min ( P < 0.05). Purification of inhibitor was achieved by heat‐treatment at 90 °C for 10 min, followed by ammonium sulphate precipitation with 30–65% saturation and size exclusion chromatography on Sephacryl S‐200, presenting a yield and purification of 53.9% and 10.91‐fold, respectively. The apparent molecular weight of trypsin inhibitor was estimated to be 14 kDa based on SDS‐PAGE and inhibitor activity of zones separated by electrophoresis. The purified inhibitor was stable over a broad pH range and retained high inhibitory activity toward trypsin after incubation at 90 °C for 60 min. NaCl, at 0–3% concentration, did not affect the inhibitory activity of purified trypsin inhibitor, however, the activity was lost when sample was treated with β‐mercaptoethanol prior to electrophoresis.