## 1 Introduction  
Lacase is a high potential oxidative enzyme with broad substrate specificity towards aromatic compounds, which makes it a promising candidate for the degradation of xenobiotics containing hydroxyl and amine groups [1; 2; 3]. However, the relatively low stability of the free enzyme arises as a major technical hurdle that hampers its large-scale application [4]. Beyond the potentiality of protein engineering and directed evolution to change enzyme conformation [5], enzyme immobilization can be applied to enhance the protein stability by the prevention of autolysis or proteolysis, rigidification of the enzyme structure via multipoint covalent attachment, and generation of favorable microenvironments [6; 7; 8]. This method has been demonstrated to improve the activity and stability of the biocatalyst in both aqueous and organic phases, provided that the support permits the diffusion of the substrate to the active site of the enzyme [9]. Furthermore, it may facilitate the simple recovery of the enzymes by centrifugation, sedimentation, or other physical separation methods and reuse in continuous systems. However, immobilized enzymes can also encounter several drawbacks, such as mass transfer limitations or interaction between the enzyme and the support that may reduce its catalytic potential [10]. Conceptually, there are two basic methods for enzyme immobilization, as the enzyme-support link can take place by physical or chemical interactions. Physical coupling methods include the entrapment of the enzyme within a tridimensional matrix, its encapsulation in an organic or inorganic polymer, and its adsorption to the support surface by ionic exchange [11], whereas covalent bonding assures the irreversible binding of the enzyme to the support matrix. Among a wide range of alternatives, the large specific surface area characteristic of nanomaterials makes this type of support an ideal candidate for enzyme immobilization [12]. The efficiency of ionic exchange depends on the pH and ionic strength of the medium as well as the hydrophobic nature of the nanoparticle surface [13; 14; 15]. Regarding covalent bonding, nanoparticles may provide a homogeneous core-shell structure, which can be functionalized to react with nucleophilic groups on the enzyme [16]. Most enzymes are covalently attached to the lysine amino groups, which are typically present on the protein surface [17]. Several factors, including pH, ionic strength, protein concentration, additives, and nanoparticles structure (porous or non-porous material) may affect the biocatalyst and the effectiveness of covalent bonding between the enzyme and the support [16; 18]. The immobilization of lactose on different types of nanoparticles such as silver and gold nanoparticles [19], chitosan-coated magnetic nanoparticles [20], and carbon nanotubes [21] has been demonstrated in recent years, although few processes have been used for practical applications at full-scale [22]. The main aim of this work is to perform the efficient immobilization of lactose on different types of magnetic and non-magnetic nanoparticles. Two different immobilization procedures will be followed: ionic exchange between the enzyme and the nanoparticle, and covalent bonding of the enzyme protein to the surface of the nanoparticle using glutaraldehyde or carbodiimide as cross-linkers [23; 24]. Glutaraldehyde, a bifunctional and versatile agent, may react with different enzyme moieties, principally involving primary amino groups of proteins, although it may eventually react with other groups such as thiols, phenols, and imidazoles [25]. On the other hand, carbodiimide is used to form amide linkage between carboxylates and amino terminal groups from the enzyme [26]. The catalytic activity of the different nanobiocatalysts will be evaluated in terms of the biotransformation potential of phenol as the model compound.