Introduction Enzymes have higher selectivity

Introduction Enzymes have higher selectivity, specificity and efficiency than chemical catalysts. Due to their properties and their green chemistry, biocatalysts are widely used in food, textile and pharmaceutical industry [1], [2]. A high efficient biocatalyst for industrial applications must be stable in a wide range of temperatures and pHs and has to be easily separated and recovered from the product during the downstream process [3], [4], [5]. Enzymes present thermal instability, susceptibility to attack by protease, activity inhibition, high sensitivity to pH and other denaturing agents, and cannot be separated at the end of reaction and reused. The immobilization of enzymes on solid supports, such as polymeric resins and inorganic materials [6], overcomes these drawbacks. In this context, nanoparticles (NPs), and in particular iron oxide NPs, have been used for the conjugation of enzymes thanks to their high surface area, high surface-volume ratio, low mass transfer limitation and their unique magnetic properties [7], [8]. Indeed, iron oxide NPs can be manipulated by an external magnetic field [9], [10], and, as a consequence of Neel and Brown relaxation, they can produce heat under alternating magnetic field (AMF), a phenomenon known as magnetic hyperthermia [10]. This has found application in biomedicine, where its use for cancer therapy and for controlling drug delivery is being extensively explored [11], [12], [13]. However, the use of this property to improve biocatalytic processes is still almost totally unexplored. Some authors, rather than using magnetic NPs as heat sources, have enhanced the activity of the linked enzymes by applying low frequency AMF. Magnetic energy is converted into a rotational motion of the enzyme-particle system that increase the collision rate with the substrate [14], [15], or triggers conformational changes on the enzyme three-dimensional structure [16]. The use of heat generated by magnetic NPs to regulate enzyme activity has been also reported, but limited to deswelling-swelling of thermosensitive polymers attached to the NP surface that force to interact substrate-bound therapeutic drugs with enzymes that trigger their release [17], [18], [19]. The effect of the heat generated by high frequencies of AMF on enzymes directly attached to NPs has been though scarcely studied. Only Suzuki and colleagues have recently reported the specific activation of α-amylase and chir99021 immobilized on ferromagnetic microparticles triggered by AMF [5], [20]. However, this effect has not been reported yet using superparamagnetic NPs, nor it has been studied the effect of the enzyme orientation on the NP surface and of conformational changes caused by the conjugation strategy. In the case of ferromagnetic microparticles, the application of a magnetic field triggers their aggregation that cannot be easily reversed since it would be necessary to heat the particles above their Curie temperature (858°K for iron oxide) [21]. Instead, in the case of superparamagnetic NPs, magnetic properties do not persist when the external magnetic field is removed. This is an important advantage of superparamagnetic NPs over ferromagnetic ones thinking on the reuse of the nanobiocatalyst. Here, we showed not only that it is possible to use AMF to activate thermophilic enzymes conjugated to superparamagnetic NPs, but also that the orientation of the enzyme molecule onto the NP surface is critical to maximize this effect. To this aim, we have functionalized iron oxide NPs with two enzymes of potential interest for industrial applications, i.e., α-amylase (AMY) from Bacillus licheniformis ( 100 °C), and l-aspartate oxidase (LASPO) from Solfolobus tokodaii ( 70 °C). We have taken into account their three-dimensional structure to conjugate them to iron oxide NPs through different native or genetically introduced residues to obtain different orientations of the enzymes. After studying the effect of the selected immobilization methodologies on the immobilization yield and activity of the bound enzyme, we carried out a physical–chemical characterization of the nano-conjugates, and applied AMF varying its frequency. We have been able to successfully activate both conjugated enzymes by the heat locally generated from the iron oxide NPs (hot-spots), without causing a significant increase in the temperature of the medium. Besides, we clearly showed that selecting an adequate immobilization methodology is a critical aspect that need to be taken into account. Indeed, the efficiency of remote enzyme activation by nanoactuation can be maximized by a specific orientation of each enzyme onto the NP surface and minimizing undesired conformational changes. Moreover, we have shown that heat remains localized around the NP-enzyme systems allowing other non-thermophilic enzymes to work together with the thermophilic ones. To this aim, we have used the non-thermophilic enzyme d-amino acid oxidase from Rhodotorula gracilis (Topt = 37 °C) in the same reaction pot of NP-AMY.