4.3.2. Metal-Based Porous Matrix

Nanoporous gold (NPG) can be obtained by acidic treatment of alloys containing 20–50% gold, or by electrochemical treatment (dealloying process). NPG structure presents interconnected ligaments and pores of width between 10 and several hundred nm. NPG displays various surface curvatures that offer a different environment for enzyme immobilization. Different enzymes have been immobilized in NPG for various purposes

showing a pore size-dependent enhanced stability as compared to enzyme in solution [144]. Alternatively, enzymes can be encapsulated in gold nanocages within a 3D gold network obtained by in situ reduction of gold salts in the presence of the enzyme [145]. Complexes formed between cations such as Cu(II) and protein molecules can serve as nucleation sites for micrometer-size particles with unique flower petal-like morphology. Enzymes confined in such structures exhibit enhanced stability [146]. Other hierarchical materials presenting mesoporous structures can be obtained by using networks of CNT or nanofibers [19].

Polymeric metal-organic-frameworks (MOFs) are matrices currently under great consideration. They consist of metal ions or clusters coordinated to organic ligands to form two- or three-dimensional structures. Cavities of mesoporous MOFs are greatly suitable for enzyme immobilization. In addition to enhanced enzyme loading and reduced leaching, the strictly controlled pore size can provide selectivity for substrates. Enzymes can be immobilized by infiltration process, requiring MOF pore size larger than protein size. Enzymes can alternatively be encapsulated in the lattices of the MOF structure by mixing the enzyme with the metal and the ligand. In this case, encapsulation proceeds through a nucleation mechanism where the enzyme acts as a nucleus for MOF growth, leading to enzymes protected in pores with size close to the radius of gyration of the protein [147]. However, in the latter case, synthesis conditions must be mild enough to avoid protein denaturation. It is admitted that the framework around the enzyme maintains the conformation of the active enzyme species [148]. Many studies report increased stability of various enzymes when embedded in the pores of MOFs. Enhanced thermal and pH stability was observed for different enzymes, as well as protection against inhibitors, or maintaining of activity in non-aqueous solvents [149–154]. MOF can also be used to encapsulate cascade of enzymes with enhanced stability and spatial control of the proteins inside the lattices [155]. However, care should be taken with solubility of MOFs in the presence of various compounds such as amino acids, some organic acids or buffers that present high affinity for the MOF-metal, inducing leaching of the enzyme via MOF dissolution [156,157]. Covalent organic frameworks, only composed of light elements (H, C, B, N, and O) and covalent bonds, with higher stability than MOFs, can be alternatives as efficient matrices for enzymes [158].

In MOFs, enzymes are statistically entrapped. It should be even more elegant to encage each individual enzyme with well-defined protecting shell surrounding it [159]. This strategy, known as single enzyme nanoparticles (SEN), is based on the controlled formation of a shell of polymer around one enzyme by in situ polymerization from the enzyme surface. The shell is thin and permeable so that substrates can freely diffuse to the enzyme core. SEN strategy is reported to enhance thermal stability, organic solvent, and acid/base tolerance even in aggressive environments [160,161]. Both protection by biocompatible polymer shell and multipoint covalent attachments within the nanocapsule were suggested to explain the enhanced enzyme stability. This last strategy recalls less recent examples reported in Section 2 of this review and based on polymer protection around the enzyme.
