In vitro Production of Hemin-Based Artificial Metalloenzymes.

Developing enzyme alternatives is pivotal to improving and enabling new processes in biotechnology and industry. Artificial metalloenzymes (ArMs) are combinations of protein scaffolds with metal elements, such as metal nanoclusters or metal-containing molecules with specific catalytic properties, which can be customized. Here, we engineered an ArM based on the consensus tetratricopeptide repeat (CTPR) scaffold by introducing a unique histidine residue to coordinate the hemin cofactor. Our results show that this engineered system exhibits robust peroxidase-like catalytic activity driven by the hemin. The expression of the scaffold and subsequent coordination of hemin was achieved by recombinant expression in bulk and through in vitro transcription and translation systems in water-in-oil drops. The ability to synthesize this system in emulsio paves the way to improve its properties by means of droplet microfluidic screenings, facilitating the exploration of the protein combinatorial space to discover improved or novel catalytic activities.

Curcumin nanocrystals-in-nanofibres as a promising platform for the management of periodontal disease.

It is estimated that nearly a half of the world's population over 30 years old suffer from some kind of periodontal disease (PD). Although preventable, PD can pose a significant health burden to patients, causing from pain and discomfort to disfigurement and death. The management of PD often requires surgical procedures accompanied of systemic antibiotic and anti-inflammatory treatments. Curcumin (CUR), a potent anti-inflammatory and antimicrobial active, has shown great promise in the management of PD; however, its effects are often limited by its low bioavailability. In this work, we report the development of electrospun nanofibres (NFs) loaded with CUR nanocrystals (NCs) for the management of PD. NCs of 100 nm were obtained by media milling and loaded into dissolving polyvinyl alcohol NFs using electrospinning. The resultant NCs-in-NFs dissolved in water spontaneously, releasing NCs with a particle size of âˆ¼120 nm. The physiochemical characterisation of the systems indicated the absence of chemical interactions between drug and polymer, and nanofibres with an amorphous nature. In vitro release profiles demonstrated that the NCs had a significantly higher dissolution rate (∼100 % at day 40) than the control group (approximately 6 % at day 40), which consisted of NFs containing a physical mixture of the drug and stabiliser. Finally, mucosal deposition studies demonstrated a 10-fold higher capacity of the novel NCs-in-NFs system to deposit CUR ex vivo using excised neonatal porcine mucosal tissue, when compared to the control group.

A Versatile Chemoenzymatic Nanoreactor that Mimics NAD(P)H Oxidase for the In Situ Regeneration of Cofactors.

Herein, we report a multifunctional chemoenzymatic nanoreactor (NanoNOx) for the glucose-controlled regeneration of natural and artificial nicotinamide cofactors. NanoNOx are built of glucose oxidase-polymer 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 NADH-dependent 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 one-pot cascade reactions.

Metal-Organic Enzyme Nanogels as Nanointegrated Self-Reporting Chemobiosensors.

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.

Tunable Polymeric Scaffolds for Enzyme Immobilization.

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.