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  • Some authors rather than using magnetic NPs as


    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 nelfinavir 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.
    Results and discussion
    Conclusions In this work, we have conjugated two thermophilic enzymes (i.e., AMY and LASPO) to iron oxide NPs through different conjugation strategies obtaining efficient biocatalysts. We have demonstrated that these NP-enzyme systems can be successfully activated by an AMF in a “wireless” fashion. We have also shown that, notwithstanding the AMF activation, the temperature of the medium increases only slightly (Fig. 10). Indeed, non-thermophilic enzymes are able to work in the same pot with the NP-AMY systems. These results made us think that there is plenty of space among the suspended NP-enzyme systems, where a non-thermophilic enzyme can work at its optimal temperature.