In the search for new biotechnological advances, increasing attention is currently being paid to the development of magnetic nanoplatforms loaded with enzymes, since, on the one hand, they can be recovered and reused, and on the other hand, they improve their catalytic activity and increase their stability, avoiding processes such as aggregation or autolysis. In this review, we evaluate a series of key parameters governing the enzyme–nanoparticle immobilization phenomena from a thermodynamic and kinetic point of view. We also focus on the use of magnetite nanoparticles (MNPs) as multifunctional vectors able to anchor enzymes, summarize the most relevant aspects of functionalization and immobilization and, finally, describe some recent and relevant applications of the enzyme–MNP hybrids as biocatalysts with especial emphasis on cancer therapy.

The bacterial phosphoenolpyruvate-dependent sugar phosphotransferase system is a multiprotein complex that phosphorylates and, concomitantly, transports carbohydrates across the membrane into the cell. The first protein of the cascade is a multidomain protein so-called enzyme I (EI). The N-terminal domain of EI from Streptomyces coelicolor, EIN^sc, responsible for the binding to the second protein in the cascade (the histidine phosphocarrier, HPr), was cloned and successfully expressed and purified. We have previously shown that EI^sc binds to HPr^sc with smaller affinity than other members of the EI and HPr families [Hurtado-Gómez et al. (2008) Biophys. J., 95, 1336–1348]. We think that the study of the isolated binding HPr^sc domain, that is EIN^sc, could shed light on the small affinity value measured. Therefore, in this work we present a detailed description of the structural features of the EIN domain, as a first step towards a complete characterization of the molecular recognition process between the two proteins. We show that EIN^sc is a folded protein, with α-helix and β-sheet structures and also random-coil conformations, as shown by circular dichroism (CD), FTIR and NMR spectroscopies. The acquisition of secondary and tertiary structures, and the burial of hydrophobic regions, occurred concomitantly at acidic pHs, but at very low pH, the domain acquired a molten-globule conformation. The EIN^sc protein was not very stable, with an apparent conformational free energy change upon unfolding, ΔG, of 4.1 ± 0.4 kcal mol⁻¹, which was pH independent in the range explored (from pH 6.0 to 8.5). The thermal denaturation midpoint, which was also pH invariant, was similar to that measured in the isolated intact EI^sc. Although EIN^sc shows thermal- and chemical denaturations that seems to follow a two-state mechanism, there is evidence of residual structure in the chemical and thermally unfolded states, as indicated by differential scanning calorimetry and CD measurements.

Eutectogels are advanced gel-based systems that integrate deep eutectic solvents (DES) into polymer networks. In this study, we report the first detailed characterization of an enzyme-containing eutectogel, representing a significant step toward advanced biosensing and biocatalytic applications. Specifically, we have incorporated pancreatic lipase, one of the main target enzymes in the treatment of obesity, in eutectogels via UV-induced radical polymerization of suitable precursors in appropriate DESs. Prior to immobilization, the enzyme was solubilized in selected DESs and its activity and conformational stability were evaluated using colorimetry and intrinsic fluorescence. Combinations of choline chloride/glycerol and tetramethylammonium chloride/glycerol were shown to be effective media for preserving and enhancing enzymatic function and conformational stability. The enzyme immersed in eutectogel exhibited high structural integrity and excellent thermal stability, maintaining its activity over several weeks. The ability of this new material to screen enzyme inhibitors was assessed using orlistat, a well-established anti-obesity agent. The results demonstrated clear detection of the drug’s inhibitory effect, even at nanomolar concentrations, highlighting the material’s potential as a screening platform for novel inhibitors with prospective anti-obesity activity. Furthermore, the device proved effective in quantifying drug presence, offering a promising and highly sensitive tool for pharmaceutical quality control applications.