Stability and activity of immobilized trypsin on carboxymethyl chitosan-functionalized magnetic nanoparticles...

Enzyme cross-linkers, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde (GA), have been used to improve the stability of immobilized enzymes. We have developed a relatively stable and high-activity immobilized trypsin through EDC and GA cross-linking. Carboxymethyl chitosan (CM-CTS)-functionalized magnetic nanoparticles (Fe₃O₄@CM-CTS) were prepared, and characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis, X-ray diffraction, and transmission electron microscopy. The CM-CTS loading of Fe₃O₄ @CM-CTS was 8.701%, and the CM-CTS coating did not cause any significant changes in the crystal structure of Fe₃O₄. The Fe₃O₄@CM-CTS nanoparticles exhibited superparamagnetic properties. Trypsin was successfully immobilized on Fe₃O₄@CM-CTS via EDC and GA cross-linking (Fe₃O₄@CM-CTS-EDC-TRY and Fe₃O₄@CM-CTS-GA-TRY, respectively). Trypsin immobilization was verified by FTIR and enzyme assays. Changes in the secondary structures of the immobilized trypsin were present in both Fe₃O₄@CM-CTS-EDC-TRY and Fe₃O₄@CM-CTS-GA-TRY. However, kinetic studies demonstrated that the immobilized trypsin retained efficient biocatalytic activity. Fe₃O₄@CM-CTS-EDC-TRY and Fe₃O₄@CM-CTS-GA-TRY both showed maximum catalytic activity at pH 8.4 and 45°C, and retained 71% and 88.5%, respectively, of their initial activities after 6 usage cycles, and 80% and 88% of their initial activities after being stored for 14 d at 4°C. The Fe₃O₄@CM-CTS-GA-TRY showed higher activity and conformational stability than Fe₃O₄@CM-CTS-EDC-TRY, which indicates that GA is effective for the immobilization of trypsin on Fe₃O₄@CM-CTS.