Stabilization of α-chymotrypsin by covalent immobilization on amine-functionalized superparamagnetic nanogel

Stabilization of α-chymotrypsin (CT) by covalent immobilization on the amine-functionalized magnetic nanogel was studied. The amino groups containing superparamagnetic nanogel was obtained by Hoffman degradation of the polyacrylamide (PAM)-coated Fe3O4 nanoparticles prepared by facile photochemical in situ polymerization. CT was then covalently bound to the magnetic nanogel with reactive amino groups by using 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide as coupling reagent. The binding capacity was determined to be 61 mg enzyme/g nanogel by BCA protein assay. Specific activity of the immobilized CT was measured to be 0.93 U/(mg min), 59.3% as that of free CT. The obtained immobilized enzyme had better resistance to temperature and pH inactivation in comparison to free enzyme and thus widened the ranges of reaction pH and temperature. The immobilized enzyme exhibited good thermostability, storage stability and reusability. Kinetic parameters were determined for both the immobilized and free enzyme. The value of Km of the immobilized enzyme was larger than did the free form, whereas the Vmax was smaller for the immobilized enzyme.