Immobilization of Agarase from Marine Vibrio Onto Carboxyl-Functioned Magnetic Nanoparticles

A novel and efficient strategy for agarase immobilization was developed with carboxyl-functioned magnetic nanoparticles as supports. The carboxyl-functioned magnetic Fe3O4 nanoparticles (CMNPs) were first prepared by co-precipitation before agarase was covalently immobilized onto the nanoparticles, with glutaraldehyde as the activation agent. The binding of agarase onto the surface of CMNPs were then characterized by Fourier-transform infrared spectroscopy, which indicated that agarase had been successfully immobilized onto the supports. The optimal immobilization conditions for 20 mg of the carriers included 4% (v/v) glutaraldehyde, a cross-linking time of 2 h, an immobilization time of 1 h, an immobilization temperature of 4 degrees C, and 850 U of agarase. The properties of immobilized agarase were compared with those of free agarase to assess the feasibility of utilizing the immobilized enzyme for oligosaccharide preparation. The immobilized agarase showed maximal catalytic activity at pH 7.0 and 40 degrees C. After immobilization, the agarase exhibited better thermal and pH stability, as well as good operation stability. The immobilized agarase maintained approximately 40% of its initial activity even after being reused seven times. The Km (Michaelis-Menten constant) values for immobilized and free agarase were 6.9 and 11.3 mg/mL, respectively.