Nuevas enzimas termoestables aplicadas a la síntesis de nucleósidos farmacológicamente activos

Resumen

In the long-running history of biocatalysis, the last decades have seen an increasing interest in the use of enzymes in substitution for traditional chemical processes. Enzymes bring notable advantages, such as higher specificity and efficiency in bioconversions due to their regio- and stereoselectivity. Also, since they allow eliminating organic solvents and other contaminants, their use lessens the environmental impact of the reactions, a critical subject in a human society aiming for sustainability. In this regard, great efforts have been made in widening the array of enzymes able to perform either new enzymatic reactions or enhance previously available ones. During the last years, thermophilic microorganisms have received a special attention as sources for new biocatalyst. This is due to the increased thermostability and resistance to denaturation they offer in comparison with mesophilic enzymes, which allows more flexible conditions and longer half lives when used at an industrial scale. The aim of this Thesis is the identification of new biotechnologically interesting enzymatic activities in thermophilic microorganisms, with the aim of overproducing and characterizating a number of these enzymes for their application in a biocatalytical process. Several test reactions were performed on a set of eleven strains of Thermus thermophilus. The reaction of synthesis of nucleosides analogues by transglycosilation was considered particularly promising. Three novel nucleosides phosphorylases from T. thermophilus HB27 were identified as the enzymes involved in the transglycosilation. These enzymes were cloned and characterized in regards to their specificity, kinetic properties and quaternary structure. The reaction parameters were optimized. The enzymes showed remarkable properties derived from their thermophilic nature, such as a wide range of optimal pH and activity beyond water-boiling point. We described for the first time the synthesis of compounds with therapeutical properties, using a thermophilic nucleoside phosphorylase at temperatures up to 80¿C. After biochemical characterization, the selectivity against arabinose nucleosides of one of the enzymes was enhanced in a 30% by Directed Evolution. Finally, the enzymes were immobilized onto solid supports, obtaining derivates that retained the enzymatic activity while enhancing their natural thermostability. As a result of our work, three new thermostable nucleoside phosphorylases, active in the synthesis of derivates of 2,6-diaminopurine, 5-halouracil or arabinose nucleosides, currently applied in the treatment of several human diseases, are described. These enzymes show enhanced properties compared to currently available mesophilic nucleosides phosphorylases. Our results involve the enhancement of the production of biotechnologically important enzymes that will permit a higher efficient synthesis of therapeutically relevant compounds at a reduced cost and hence a broader availability for the increased global demand for new drugs and treatments.