Rhamnulosa-1-fosfato aldolasa de thermotoga maritimaCaracterización y aplicabilidad de un nuevo biocatalizador termoestable para la formación de enlaces c-c

Laburpena

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