Characterization of lactase-conjugated magnetic nanoparticles

Conjugation of lactase to magnetic nanoparticles is of interest in biosensor and ingredient processing applications that require high enzyme concentration and catalyst separation from the reaction stream. However, little is known about the effects of these materials on the physicochemical attributes of conjugated lactase. Lactase (Aspergillus oryzae) was covalently attached by carbodiimide chemistry to carboxylic-acid functionalized magnetic particles having a hydrodynamic radius of 18 nm. The resulting enzyme–nanoparticle conjugates were characterized with regard to particle size, zeta potential, enzyme kinetics, temperature and pH stability, catalyst recovery, and secondary structure changes. Following attachment, the materials retained colloidal stability and individual particle characteristics with a zeta potential of ?33 mV compared to ?46 mV for the native particle. The conjugated enzyme showed no changes in secondary structure and exhibited significant catalytic activity with a catalytic efficiency of 2.8 × 10³ M^?1 s^?1 compared to 2.5 × 10³ M^?1 s^?1 for the native enzyme. Relative to the free enzyme, the conjugated enzyme was recovered for repeated use with 78% activity retained after five cycles. This work demonstrates that carboxylic-acid functionalized magnetic nanoparticles can be utilized as a means of producing a simple and effective conjugated-lactase system that achieves both particle and enzyme stability.