BACKGROUNDHybrid enzyme-nanoparticle complexes, obtained by enzyme immobilization onto superparamagnetic particles, display unique properties for use as biocatalysts. A novel methodology for the immobilization of chloroperoxidase (CPO) onto magnetic nanoparticle clusters (mNC) is presented. Chloroperoxidase catalyzes alcohol oxidations using peroxides, and it has recently been shown to recognize -amino alcohols as substrates, although high required peroxide concentration led to poor CPO stability. RESULTSmNC retains the superparamagnetic properties of the single nanoparticle plus an increased magnetic moment, necessary for effective magnetic recovery. Different functional groups have been introduced on the silica layer that covers mNC. The linkage enzyme-support has been intended through different reactive groups on the CPO surface. The selected biocatalyst (95% yield, 63% retained activity), obtained by prior enzyme oxidation followed by coupling to the amino groups on the mNC surface, has been compared with soluble CPO in the model reaction (N-Cbz-3-aminopropanol oxidation) with significantly higher substrate conversion due to 4-fold increased enzyme stability. CONCLUSIONSFunctionalized mNC has demonstrated to be efficient for the preparation of hybrid enzyme-mNC biocatalysts. The systematic study of chloroperoxidase immobilization onto mNC led to several useful biocatalysts. The described methodology could easily be extended to many other enzymes in the preparation of efficient and reusable biocatalysts. (c) 2017 Society of Chemical Industry