Covalent-bonded immobilization of enzyme on hydrophilic polymer covering magnetic nanogels

A new mesoporous support, hydrophilic polymer covering magnetic nanogels containing reactive carboxyl groups, was obtained by photochemical in situ polymerization. The enzyme α-chymotrypsin (CT), a model for protein or esters hydrolysis, was anchored to the support by 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC) activation. The immobilization proof was confirmed with Fourier transform spectroscopy (FT-IR) and transmission electron microscopy (TEM) observation. The bound CT behaved superparamagnetic and exhibited excellent response to applied magnetic field. The immobilized CT had better resistance to temperature and pH inactivation in comparison to the free form, and hence widened the reaction pH and temperature regions. Thermostability and storage stability of the enzyme increased up on covalent immobilization. The immobilized enzyme retained a relative activity of 85% after 12 repeated uses. Kinetic parameters were assayed for both free and immobilized CT. The value of K_m (5.3 × 10^-4 M) for the immobilized enzyme was larger than that of the free counterpart (3.7 × 10^-4 M), while the V_max (6.1 U mg^-1 enzyme) was smaller for the immobilized enzyme.