Antiviral compounds are crucial to controlling the SARS-CoV-2 pandemic. Approved drugs have been tested for their efficacy against COVID-19, and new pharmaceuticals are being developed as a complementary tool to vaccines However, there are not any effective treatment against this disease yet. In this work, a cheap and fast purification method of natural tyrosinase from Agaricus bisporus fresh mushrooms was developed in order to evaluate the potential of this enzyme as a therapeutic protein by the inhibition of SARS-CoV-2 3CLpro protease activity in vitro. Tyrosinase showed a mild inhibition of 3CLpro of around 15%. Thus, different variants of this protein were synthesized through chemical modifications, covalently binding different tailor-made glycans and peptides to the amino terminal groups of the protein. These new tyrosinase conjugates were purified and characterized by circular dichroism and fluorescence spectroscopy analyses, and their stability under different conditions. Then all these tyrosinase conjugates were tested in 3CLpro protease inhibition. From them, the conjugate between tyrosinase and dextran-aspartic acid (6kDa) polymer showed the highest inhibition, with an IC50 of 2.5 ug/ml and IC90 of 5 ug/ml, results that highlight the potential use of modified tyrosinase as a therapeutic protein and opens the possibility of developing this and other enzymes as pharmaceutical drugs against diseases.

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.