The number of methodologies for the immobilization of enzymes using polymeric supports is continuously growing due to the developments in the fields of biotechnology, polymer chemistry, and nanotechnology in the last years. Despite being excellent catalysts, enzymes are very sensitive molecules and can undergo denaturation beyond their natural environment. For overcoming this issue, polymer chemistry offers a wealth of opportunities for the successful combination of enzymes with versatile natural or synthetic polymers. The fabrication of functional, stable, and robust biocatalytic hybrid materials (nanoparticles, capsules, hydrogels, or films) has been proven advantageous for several applications such as biomedicine, organic synthesis, biosensing, and bioremediation. In this review, supported with recent examples of enzyme-protein hybrids, we provide an overview of the methods used to combine both macromolecules, as well as the future directions and the main challenges that are currently being tackled in this field.

The absence of a universal enzyme immobilization method that fulfils the needs of each biocatalytic system has boosted the development of new approaches to the fabrication of heterogeneous biocatalysts. Herein, we present a protocol for the synthesis of a novel sort of catalytically responsive hybrid biomaterials, named metal–organic enzyme aggregates (MOEAs). The formation of MOEAs is triggered by the coordination of divalent metal cations to imidazole-decorated enzyme nanogels in a fast and effective assembly mechanism. The size and morphology of MOEAs can be tailored from small individual particles to macroscopic aggregates, which are stable in water and disassemble in the presence of a complexing agent. Finally, the extensive compositional and catalytic characterization of the hybrids showed high transformation rates, significant protein loads, and great thermostability. These features revealed MOEAs as an excellent alternative as carrierless immobilization system.

Here, a platform for the development of highly responsive organicinorganic enzyme hybrids is provided. The approach entails a first step of protein engineering, in which individual enzymes are armored with a porous nanogel decorated with imidazole motifs. In a second step, by mimicking the biomineralization mechanism, the assembly of the imidazole nanogels with CuSO 4 and phosphate salts is triggered. A full characterization of the new composites reveals the first reported example in which the assembly mechanism is triggered by the sum of Cu(II)-imidazole interaction and Cu 3 (PO 4 ) 2 inorganic salt formation. It is demonstrated that the organic component of the hybrids, namely the imidazole-modified polyacrylamide hydrogel, provides a favorable spatial distribution for the enzyme. This results in enhanced conversion rates, robustness of the composite at low pH values, and a remarkable thermal stability at 65 °C, exhibiting 400% of the activity of the mineralized enzyme lacking the organic constituent. Importantly, unlike in previous works, the protocol applies to the use of a broad range of transition metal cations (including mono-, di-, and trivalent cations) to trigger the mineralization mechanism, which eventually broadens the chemical and structural diversity of organic-inorganic protein hybrids.

Nevertheless, the utilization of biomolecules, i.e., proteins, to impart functionality to inorganic and/or organic materials and afford highly efficient functional devices presents a number of challenges in the research of functional biomaterials. [4][5][6] The main limitations predominantly arise from intermolecular aggregation, surfaceinduced denaturation, steric hindrance of active sites, and lack of dynamical freedom imposed by solid state. [7][8][9] The deposition of continuous protein thin films seems to be a good strategy that fulfill those needs. [10,11] With particular emphasis on biocatalytic coatings, the fabrication method should guarantee high enzyme loads, low substrate/product flow transport limitations, and improve the lifetime and stability of the biomolecule. [12] Currently, reported methods for the fabrication of functional biofilms are based on the utilization of a relatively limited range of naturally self-assembling proteins, layer-by-layer deposition approaches, and the adsorption of the proteins to amphiphilic copolymers. [13][14][15][16][17] However, these approaches usually require of the covalent crosslinking of the components in order to avoid the disaggregation of the film in water and at broad range of pH. [18] Yet, the uncontrolled covalent crosslinking might be especially damaging in the formation of functional protein films. The protein's amino acids can be altered and severe substrate diffusion issues might be caused within the film, resulting in impaired biomaterials. [19] Therefore, an alternative sequence-independent methodology that allows the fabrication of functional protein films would vastly expand the toolkit for creating biomaterials.In this regard, the bioinspired self-assembly of hierarchically structured peptide or protein films is an attractive approach. [20][21][22][23] In nature, the metal-driven crosslinking of specific peptidic building blocks leads to complex hierarchical structures across many lengths, as it happens in mussel byssus or worm jaws. [24][25][26] Furthermore, metal-directed protein selfassembly (MDPSA) methodology is inspired by the affinity of distinct residue side chains such as histidines, cysteines, lysines, and asparagines toward metallic cations (mainly Ni, Cu, Co, and Zn). MDPSA allocates such key residues on the surface of the protein as anchoring points. [27][28][29] Hence, metal ions are used as inorganic bridges that not only guide the assembly of the proteins into hierarchical architectures, but also might The deposition of protein thin films on (in)organic surfaces is a key approach to incorporate new functionalities into these materials for a broad number of applications. However, most of the current methods used for the controlled assembly of such biomolecules and eventual film formation are limiting since entail either the chemical modification of the proteins, which leads sometimes to impaired materials, or the sequential layer-by-layer deposition of charged macromolecules. In this work, a facile bioinspired method for the versati...

Herein, the design, synthesis, and characterization of bifunctional hybrid nanoreactors used for concurrent one‐pot chemoenzymatic reactions are shown. In the design, the enzyme, glucose oxidase, is wrapped with a peroxidase‐mimetic catalytic polymer. Hemin, the organic catalyst, is linked to the flexible polymeric scaffold through coordination to the imidazole groups that hang out the network. This spatial arrangement, which works as a metabolic channel, is optimized for cooperative chemoenzymatic reactions in which the enzyme catalyzes first. A deep characterization of the integrated nanoreactors demonstrates that the confinement of two distinct catalytic sites in the nanospace is very effective in one‐pot reactions. Moreover, besides its role as scaffold material, the polymeric mantel protects both the biocatalyst and the chemical catalyst from degradation and inactivation in the presence of organic solvents. Furthermore, the polymeric environment of the nanoreactors can be tailored in order to trigger the assembly of those into highly active heterogeneous hybrid catalysts. Finally, the new nanoreactors are applied to the efficient degradation of organic aromatic compounds using glucose as the only fuel.

A fluorometric glucose biosensor based on fine-tuned chemoenzymatic nanohybrids is herein proposed. The successful integration of an engineered glucose oxidase enzyme and an optically responsive polymeric nanogel in a single entity has led to the fabrication of a highly efficient glucose chemobiosensor. The optical responsiveness has been achieved by the loading of preactivated polymeric hydrogel with fluorescent lanthanide, i.e., cerium (III), cations. A comprehensive investigation of the responsiveness of the biomaterial revealed the interplay between the oxidation state of the cerium lanthanide and the fluorescence emission of the polymer. Finally, a full structural, chemical, and biochemical characterization of the reported system supports the chemobiosensors as robust, specific, and sensitive materials that could be utilized to faithfully quantify the amount of glucose in tear fluids.

Herein, we report a multifunctional chemoenzymatic nanoreactor (NanoNOx) for the glucosecontrolled regeneration of natural and artificial nicotinamide cofactors. NanoNOx are built of glucose oxidasepolymer hybrids that assemble in the presence of an organometallic catalyst: hemin. The design of the hybrid is optimized to increase the effectiveness and the directional channeling at low substrate concentration. Importantly, NanoNOx can be reutilized without affecting the catalytic properties, can show high stability in the presence of organic solvents, and can effectively oxidize assorted natural and artificial enzyme cofactors. Finally, the hybrid was successfully coupled with NADHdependent dehydrogenases in one-pot reactions, using a strategy based on the sequential injection of a fuel, namely, glucose. Hence, this study describes the first example of a hybrid chemoenzymatic nanomaterial able to efficiently mimic NOx enzymes in cooperative onepot cascade reactions.

Ein neuartiges Hybridmaterial, das die NAD(P)H‐Oxidase nachahmt, wurde für die In‐situ‐Regeneration verschiedener Enzym‐Cofaktoren getestet, wie Ana Beloqui et al. in ihrer Zuschrift berichten (e202206926). Dieses Material wurde durch die kontrollierte Assemblierung von Glukoseoxidase‐ und Hemin‐Katalysatoren auf einem funktionellen Nanogel hergestellt, das die (Bio‐)Katalysatoren in verschiedene Kompartimente anordnet. Das Syntheseverfahren ermöglicht die Herstellung von heterogenen Nanoreaktoren, die sich als sehr effektiv für die In‐situ‐Regeneration natürlicher und künstlicher Enzym‐Cofaktoren erwiesen haben.