Preparation of Superparamagnetic Fe₃O₄@Alginate/Chitosan Nanospheres for Candida rugosa lipase Immobilization and Utilization of Layer-by-Layer Assembly to Enhance the Stability of Immobilized Lipase

Superparamagnetic alginate nanospheres with diameter of 50 nm were prepared by self-assembly of alginate in the Ca(2+) solution; and then superparamagnetic alginate/chitosan nanospheres, which have positive charge and could adsorb lipase directly, were obtained with a following assembly of chitosan based on the electrostatic interaction between alginate and chitosan. Subsequently, oxidic poly (ethylene glycol) was used to functionalize the magnetic alginate/chitosan nanospheres. Thus, the magnetic nanospheres with aldehyde groups and a brushlike structure were formed. With various characterizations, it was verified that the magnetic alginate/chitosan nanospheres held small diameters (around 60 nm) and displayed superparamagnetism with high saturation magnetization. The Candida rugosa lipase (CRL), meanwhile, was immobilized onto the magnetic alginate/chitosan nanospheres by electrostatic adsorption and covalent bonding, respectively. Afterward, a layer-by-layer (LBL) assembly process was utilized to coat the immobilized CRL (ICRL) with covering layers made up of alginate and chitosan. After studying the properties of ICRL such as activity, kinetic behaviors, stability and reusability, it was proved that the ICRL prepared with two methods displayed more excellent properties than that prepared with electrostatic adsorption only. Additionally, coating ICRL with covering layers showed good effect on improving the stability of ICRL.