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Nuevos sistemas biocatalíticos para la formación de enlaces C-C. Diseño, caracterización y aplicación sintética de una enzima de fusión quinasa-aldolasa

AuthorsIturrate Montoya, Laura
AdvisorGarcía-Junceda, Eduardo
Enzimas de fusión
Sistemas multi-enzimáticos
Formación enlaces C-C
Issue Date2008
PublisherUniversidad Autónoma de Madrid
AbstractThe utility of a multienzyme system for C-C bond formation catalysed by dihydroxyacetone phosphate-dependent aldolases employing aldehydes with great structural diversity as acceptor substrates has been studied. This system was composed by recombinant dihydroxyacetone kinase from Citrobacter freundii CECT 4626 for in situ dihydroxyacetone phosphate formation, two synthetically useful dihydroxyacetone phosphate-dependent aldolases —Lfuculose- 1-phosphate aldolase and L-rhamnulose-1-phosphate aldolase— and acetate kinase for the ATP regeneration. Two different one-pot strategies were developed in order to adapt the multienzyme system to the reaction conditions showing the great feasibility of the biocatalytic system. With the aim of simplifying this multienzyme system a new bi-functional fusion protein has been constructed by gene fusion by overlap extension. This fusion enzyme consists of monomeric fructose-1,6-biphosphate aldolase from Staphylococcus carnosus and the homodimeric dihydroxyacetone phosphate kinase from C. freundii CECT 4626 with an intervening five aminoacids linker. The fusion protein was expressed soluble and retained both kinase and aldolase activity. The secondary structure of the bi-functional enzyme has been analyzed by CD spectroscopy, as well as the parental enzymes, in order to study the effect of the covalent union of the two parents proteins on the structure and thermal stability of the fused enzyme. In addition, the kinetic parameters were studied for both fusion and parent enzymes, showing no effect of fusion on the KM values while the kcat values were smaller for the fusion protein than those from the parental partners. The kcat value for the kinase activity in the fusion enzyme (3.53 x 102) was about one order of magnitude lower than the kcat of its native equivalent and in the case of the aldolase activity the kcat (1.70 x 103) drop 4 orders of magnitude. Besides this lost in the turnover number, the fusion enzyme maintains a so good catalytic efficiency (kcat/KM) about 104 min-1x mM-1. The proximity of the active centres in the fusion enzyme promotes a kinetic advantage as indicate the 20-fold increment in the initial velocity of the overall aldol reaction. Finally, the applicability of the fusion enzyme for C-C bond formation using DHA as donor has been tested.
DescriptionTesis doctoral leida en la Universidad Autónoma de Madrid. Director de Tesis: Dr. Eduardo García-Junceda Redondo. Instituto de Química Orgánica General. CSIC Tutor: Dr. Edgardo Catalán Tobar. Realizada bajo su dirección en el Departamento de Química Orgánica Biológica del Instituto de Química Orgánica General del Consejo Superior de Investigaciones Científicas
Appears in Collections:(IQOG) Tesis
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