Preparation of trypsin-immobilised chitosan beads and their application to the purification of soybean trypsin inhibitor

BACKGROUND: Trypsin inhibitors are among the most important antinutritional factors in legumes. Recent research has shown that soybean trypsin inhibitor (SBTI) exhibits multiple bioactivities, but very few studies on the purification of SBTI are available. Enzymes are commonly used as biospecific ligands in affinity purification of their substrates or inhibitors. The aim of the present study was to prepare trypsin (EC 3.4.21.4)-immobilised chitosan beads and use them to purify trypsin inhibitor from soybean whey. RESULTS: Compared with free trypsin, the immobilised trypsin had higher thermal and pH stability. The adsorption ratio of SBTI from crude SBTI aqueous solution by trypsin-immobilised chitosan beads was 33.3%. The purified SBTI obtained by affinity chromatography was characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis as a single polypeptide band with an M-r of 8.3 kDa belonging to the Bowman-Birk family. CONCLUSION: Trypsin-immobilised chitosan beads were effectively used in the affinity separation of trypsin inhibitor from soybean seeds, thus indicating that immobilised trypsin may have practical application in the soybean-processing industry. The results of this study provide a background for further investigation of potential applications of soybean bioactive constituents in the areas of agriculture and food. (c) 2008 Society of Chemical Industry.