Enzymes Immobilized on Superparamagnetic Fe₃O₄@Clays Nanocomposites: Preparation, Characterization, and a New Strategy for the Regeneration of Supports

Abstract

We present a simple method to produce superparamagnetic Fe 3 O 4 @Clays nanocomposites consisting of magnetic iron oxide nanoparticles orderly self-assembled on some restricted positions of nanoclays. The as-prepared Fe 3 O 4 @Clays have highly ordered structure, large surface area, and high magnetic sensitivity, as verified by transmission electron microscopy (TEM), IR spectroscopy, X-ray diffraction (XRD), nitrogen adsorption−desorption isotherms, and a vibrating sample magnetometer (VSM). Subsequently, the as-prepared Fe 3 O 4 @Clays treated with (3-aminopropyl)triethoxysilane are used as immobilization material. The conjugation of glucoamylase, hereby chosen as model enzyme, onto the amino-functionalized magnetic nanoparticles by using glutaraldehyde as a coupling reagent is further demonstrated and assessed based on its activity, kinetics, and thermal stability as well as reusability. Inspired by the structural character of enzyme (containing functional residues that are ideal reaction sites for the immobilization of enzymes once more), two novel regenerated strategies of supports are successfully developed to regenerate the carriers at the end of the life of the immobilized glucoamylase. The quality of glucoamylase immobilized on the regenerated supports is also defined by determining of the enzyme activity, kinetics, thermal stability, and reusability. The results indicate that the strategies for the regeneration of supports are viable. The applicability of the regenerated strategies of supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase. The regenerated strategies also offer an attractive and flexible alternative to regenerate other traditional supports at the end of the life of the immobilized enzyme.