IMPROVEMENT OF PURIFICATION OF TRYPSIN INHIBITOR FROM WILD SOYBEAN (GLYCINE SOJA SIEB. & ZUCC.) USING CHITOSAN RESIN‐IMMOBILIZED TRYPSIN

Chitosan resin-trypsin was prepared for one-step affinity purification of trypsin inhibitor (TI) from a wild-type soybean (Glycine soja Sieb. & Zucc.). The influences of several parameters (cross-linking, chemical modifying and activating agent) on the chitosan resin-trypsin activity were investigated. The results showed that the chitosan resin-trypsin composite demonstrated considerable trypsin activity at levels of, 3.6% glutaraldehyde (cross-linking agent), 0.06 mol/L NaBH4 (chemical modifying agent) and 15% epichlorohydrin (activating agent). Furthermore, the trypsin activity assay indicated that chitosan resin-trypsin could tolerate relatively high temperature (65C) and wide pH range (5.0–9.0), compared with free trypsin (45C, pH 6.0–8.0). With chitosan resin-trypsin as matrix on affinity chromatography column, a Bowman-Birk trypsin inhibitor from a wild soybean was purified (adsorption capacity of chitosan resin-trypsin for TI was about 1.33 mg/g wet matrix), with homogeneous MW of 8.2 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The affinity chromatography purification method described for TI was 279-fold with a 58.3% recovery rate of TI, much improved over established procedures. PRACTICAL APPLICATIONS The use of chitosan resin as a support for enzymes in affinity chromatography has advantages over other materials: low cost, good stability and versatility. The covalent immobilization of trypsin on chitosan resin permits easy recovery and reuse of the enzyme, and produces a quick and effective tool to isolate the Bowman-Birk trypsin inhibitor form wild soybean. This method could be a competitive choice for large-scale purification of this functional molecule.