Artificial Manganese Metalloenzymes with Laccase-like Activity: Design, Synthesis, and Characterization.

Laccase is an oxidase of great industrial interest due to its ability to catalyze oxidation processes of phenols and persistent organic pollutants. However, it is susceptible to denaturation at high temperatures, sensitive to pH, and unstable in the presence of high concentrations of solvents, which is a issue for industrial use. To solve this problem, this work develops the synthesis in an aqueous medium of a new Mn metalloenzyme with laccase oxidase mimetic catalytic activity. Geobacillus thermocatenulatus lipase (GTL) was used as a scaffold enzyme, mixed with a manganese salt at 50 °C in an aqueous medium. This leads to the in situ formation of manganese(IV) oxide nanowires that interact with the enzyme, yielding a GTL-Mn bionanohybrid. On the other hand, its oxidative activity was evaluated using the ABTS assay, obtaining a catalytic efficiency 300 times higher than that of Trametes versicolor laccase. This new Mn metalloenzyme was 2 times more stable at 40 °C, 3 times more stable in the presence of 10% acetonitrile, and 10 times more stable in 20% acetonitrile than Novozym 51003 laccase. Furthermore, the site-selective immobilized GTL-Mn showed a much higher stability than the soluble form. The oxidase-like activity of this Mn metalloenzyme was successfully demonstrated against other substrates, such as l-DOPA or phloridzin, in oligomerization reactions.

Glyconanomaterials for Human Virus Detection and Inhibition.

Viruses are among the most infectious pathogens, responsible for the highest death toll around the world. Lack of effective clinical drugs for most viral diseases emphasizes the need for speedy and accurate diagnosis at early stages of infection to prevent rapid spread of the pathogens. Glycans are important molecules which are involved in different biological recognition processes, especially in the spread of infection by mediating virus interaction with endothelial cells. Thus, novel strategies based on nanotechnology have been developed for identifying and inhibiting viruses in a fast, selective, and precise way. The nanosized nature of nanomaterials and their exclusive optical, electronic, magnetic, and mechanical features can improve patient care through using sensors with minimal invasiveness and extreme sensitivity. This review provides an overview of the latest advances of functionalized glyconanomaterials, for rapid and selective biosensing detection of molecules as biomarkers or specific glycoproteins and as novel promising antiviral agents for different kinds of serious viruses, such as the Dengue virus, Ebola virus, influenza virus, human immunodeficiency virus (HIV), influenza virus, Zika virus, or coronavirus SARS-CoV-2 (COVID-19).

Geranyl Functionalized Materials for Site-Specific Co-Immobilization of Proteins.

Different materials containing carboxylic groups have been functionalized with geranyl-amine molecules by using an EDC/NHS strategy. Chemical modification of the support was confirmed by XRD, UV-spectrophotometer, and FT-IR. This geranyl-functionalized material was successfully applied for four different strategies of site-selective immobilization of proteins at room temperature and aqueous media. A reversible hydrophobic immobilization of proteins (lipases, phosphoglucosidases, or tyrosinase) was performed in neutral pH in yields from 40 to >99%. An increase of the activity in the case of lipases was observed from a range of 2 to 4 times with respect to the initial activity in solution. When chemically or genetically functionalized cysteine enzymes were used, the covalent immobilization, via a selective thiol-alkene reaction, was observed in the presence of geranyl support at pH 8 in lipases in the presence of detergent (to avoid the previous hydrophobic interactions). Covalent attachment was confirmed with no release of protein after immobilization by incubation with hydrophobic molecules. In the case of a selenium-containing enzyme produced by the selenomethionine pathway, the selective immobilization was successfully yielded at acidic pH (pH 5) (89%) much better than at pH 8. In addition, when an azido-enzyme was produced by the azide-homoalanine pathway, the selective immobilization was successful at pH 6 and in the presence of CuI for the click chemistry reaction.

Pd-Oxazolone complexes conjugated to an engineered enzyme: improving fluorescence and catalytic properties.

Different Pd-complexes containing orthometallated push-pull oxazolones were inserted by supramolecular Pd-amino acid coordination on two genetically engineered modified variants of the thermoalkalophilic Geobacillus thermocatenolatus lipase (GTL). Pd-lipase conjugation was performed on the solid phase in the previously immobilized form of GTL under mild conditions, and soluble conjugated Pd-GTL complexes were obtained by simply desorbing by washing with an acetonitrile aqueous solution. Three different Pd complexes were incorporated into two different genetically modified enzyme variants, one containing all the natural cysteine residues changed to serine residues, and another variant including an additional Cys mutation directly in the catalytic serine (Ser114Cys). The new Pd-enzyme conjugates were fluorescent even at ppm concentrations, while under the same conditions free Pd complexes did not show fluorescence at all. The Pd conjugation with the enzyme extremely increases the catalytic profile of the corresponding Pd complex from 200 to almost 1000-fold in the hydrogenation of arenes in aqueous media, achieving in the case of GTL conjugated with orthopalladated 4a an outstanding TOF value of 27 428 min-1. Also the applicability of GTL-C114 conjugated with orthopalladated 4b in a site-selective C-H activation reaction under mild conditions has been demonstrated. Therefore, the Pd incorporation into the enzyme produces a highly stable conjugate, and improves remarkably the catalytic activity and selectivity, as well as the fluorescence intensity, of the Pd complexes.