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IMPROVEMENT OF PURIFICATION OF TRYPSIN INHIBITOR FROM WILD SOYBEAN (GLYCINE SOJA SIEB. & ZUCC.) USING CHITOSAN RESIN‐IMMOBILIZED TRYPSIN
Author(s) -
ZHANG BIN,
ZHANG LI,
WANG DONGFENG,
SUN JIPENG
Publication year - 2011
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.2010.00489.x
Subject(s) - trypsin , chitosan , chromatography , chemistry , trypsin inhibitor , kunitz sti protease inhibitor , affinity chromatography , glycine , chemical modification , enzyme , biochemistry , amino acid
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 NaBH 4 (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.