*4.2. Nanomaterials for Stabilizing Enzyme in the Immobilized State*

Interest in nanomaterials is increasingly growing thanks to their intrinsic physicochemical properties that allow to envision many applications when combined with enzymes for energy conversion, biosensing, drug delivery, etc. The most attractive feature of nanomaterials is their large surface-to-volume ratio that enables enhanced catalysis thanks to an increased loading of enzymes. Nanomaterials are easily functionalized for further enzyme attachment via classical covalent coupling or click chemistry. However, they also present heterogeneity in terms of size of particles [124], number of sites on the surface available for enzyme attachment, or local curvature that will greatly influence enzyme immobilization. Particle agglomeration, caused by low colloidal stability of the particles in buffer, can also hamper the storage stability of the immobilized biosystems [125].

Nanoparticles (NP) are widely used in medicine, cosmetics, or food industries. Hence, the effect of enzyme immobilization on NPs on both activity and stability has been largely studied over the last ten years. Enhanced thermal or storage stability are reported, the

reasons being often related to the mode of attachment of the enzyme [126,127]. As examples, enhanced thermal and long term stabilities, resistance to urea or to acidic conditions were shown for GOx on ferritin [128] or on Fe3O4-based NPs [129], for lipase on Fe3O4-based NPs [130], for galactosidase on ZnO-NPs [131], or for cellulase on magnetic NPs [132]. The linkage to the NP was assumed to prevent unfolding of the enzyme no matter of the NP intrinsic property.

Other aspects that account for enzyme stability enhancement on NPs need to be discussed. One of the challenges is the control of the number of grafted enzymes, which may have a direct impact on enzyme flexibility, hence stability. Beyond conjugation of the enzyme to the NP that can affect aggregation, role of the charge, size, and morphology of the NP, local pH and ionic strength may be key parameters involved in the stabilization process [127]. For instance, NP size was shown to have a direct effect on the stability of the enzyme [133]. Varying the size of NPs induces the variation of the NP surface curvature, that will offer distinguished surface of contact for the enzyme. High NP surface curvature (diameters of NP less than 20 nm) was shown to preserve enzyme native conformation [134]. Smaller area for protein contact as well as suppression of unfavorable protein–protein lateral interactions are most probably the reasons that can account for stabilization effect of nanomaterials [135,136]. Curvature-based stabilization of enzymes can be extended to other nanomaterials such as carbon nanotubes (CNT) [4,135]. Morphology of the NP was also reported to tune the enzyme stability. For example, switching from nanospheres to nanorods decreases enzyme stability, most probably because of the impact of the flat cylindrical axial surface of nanorods [137].
