In this Thesis, is described the heterologous expression of rhamnulose-1-phosphate aldolase from Thermotoga maritima, —a hyperthermophilic bacterium with an optimum growth temperature of 80 °C— the biochemical characterization of the recombinant enzyme and the study of its applicability as biocatalyst. The biochemical characterization allowed us to conclude that: • Rhamnulose-1-phosphate aldolase from T. maritima is a class II DHAP-dependent aldolase, which catalyses aldol addition using Co2+. •Rha-1PA from T. maritima is a hyperthermophile enzyme with optimum activity measured at 95 °C. Furthermore, in contrast with other hyperthermophilic enzymes, it maintains a significant percentage of activity at R/T. • Rha-1PA from T. maritima can be used in a wide range of reaction conditions, including different temperatures (between 20 and 95 °C), pH values (at least 50% of activity between pH 5.7 and 9.0) and in the presence of organic co-solvents (90% of the initial activity in H2O/AcN 60:40, 100% in H2O/DMSO 80:20, 50% in H2O/DMF 75:25 and 40% in 90:10 H2O/isopropanol and H2O/THF). • The significant stability of the aldolase at high temperatures has been shown, displaying a TM of 102 °C and showing a half-life of 44, 33 and 3.5 h at 80, 95 and 115 °C, respectively. • His-tag and cofactor affect not only the secondary structure of the protein, but also its thermostability. Synthetic applicability studies allowed us to conclude that: • Rha-1PA from T. maritima is a useful biocatalyst that is compatible with multi-enzyme systems for C-C bond formation involving mesophilic enzymes. • This aldolase can be applied to the synthesis of several organic compounds such as aldols, nitrocyclitols and iminocyclitols, in which similar or higher yields than Rha-1PA from E. coli had been obtained. • Stereoselectivity of the enzyme is similar to RhA-1Pa from E. coli. In both cases, stereoselectivity control is not total, since mixtures of L-threo (R,S) and D-erythro (R,R) diastereoisomers were obtained. • The study of the stereochemistry showed that in all the cases, the major diastereoisomer formed was coincidental with the natural stereopreference of the enzyme (L-threo (R,S)), except when aldehyde 1l was used as an acceptor. • Reaction control of the enzyme was studied, being the formation of L-threo isomer kinetically controlled and D-erythro isomer under thermodynamic control

Tesis doctoral defendida en la Universidad Complutense de Madrid el 19 de septiembre de 2014. Se ha realizado en el departamento de Química Bioorgánica del Instituto de Química Orgánica General (CSIC), bajo la dirección del Dr. Eduardo García-Junceda Redondo, dentro del programa de doctorado de Química Sostenible de la Facultad de Ciencias Químicas de la Universidad Complutense de Madrid.

El trabajo ha estado financiado por el Ministerio de Ciencia e Innovación (Grant CTQ2007-67403/BQU, Grant CTQ2010-15418, Grant PI11/01436) y por la Comunidad de Madrid (CAPOTE S2009/PPQ-1752). Dña. Isabel Alicia Oroz Guinea ha sido financiada por una Beca-Contrato del Programa JAEPredoc del Consejo Superior de Investigaciones Científicas (convocatoria 2008).

Peer reviewed