Conjugation of enzyme on superparamagnetic nanogels covered with carboxyl groups

Alpha-chymotrypsin (CT) as model enzyme was conjugated onto the novel carboxyl-functionalized superparamagnetic nanogels, prepared via facile photochemical in situ polymerization, by using 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC) as coupling reagent. The obtained magnetic immobilized enzyme was characterized by use of photo correlation spectroscopy (PCS), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) measurement, thermogravimetric (TG) analysis and vibrating sample magnetometer (VSM) measurement. PCS result showed that the immobilized enzyme was 68 nm in diameter while the magnetic nanogels with carboxyl groups were only 38 nm; enzyme immobilization led to pronounced change in size. Superparamagnetic properties were retained for Fe3O4 after enzyme immobilization while slightly reducing its value of saturation magnetization. Immobilization and surface coating did not induce phase change of Fe3O4 by XRD analysis. The binding capacity was 30 mg enzyme/g and 37.5 mg enzyme/g nanogel determined by TG analysis and BCA protein assay, respectively. Specific activity of the immobilized CT was calculated to be 0.77 U/(mg min), 82.7% as that of the free form.