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dc.contributor.authorIturrate Montoya, Laura-
dc.contributor.authorSánchez-Moreno, Israel-
dc.contributor.authorOroz-Guinea, Isabel-
dc.contributor.authorPérez-Gil, Jesús-
dc.contributor.authorGarcía-Junceda, Eduardo-
dc.identifier.citationChemistry - a European Journal 16(13): 4018-4030 (2010)es_ES
dc.description“This is the pre-peer reviewed version of the following article: Iturrate, L., Sánchez-Moreno, I., Oroz-Guinea, I., Pérez-Gil, J., García-Junceda, E. (2010) “Preparation and characterization of a bifunctional aldolase/kinase enzyme. A more efficient biocatalyst for C-C bond formation“ Chem. Eur. J., 16, 4018-4030, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/chem.200903096/abstract”es_ES
dc.description.abstractA bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening five amino acid linker. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structure of the bifuctional enzyme has been analysed by CD spectroscopy, as well as that of the parental enzymes, in order to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Since S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centres in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channellinges_ES
dc.description.sponsorshipWe thank the Spanish Ministerio de Ciencia e Innovación for financial support (Grant CTQ2007-67403/BQU). J. P.-G. has been supported by grants BIO2009-09694 and CSD2007-00010. L. Iturrate and I. Sánchez-Moreno acknowledges the Predoctoral Fellowship from Comunidad de Madrid. I. Oroz-Guinea is a JAEPredoc fellow from CSIC. We thank E. G. Doyagüez for her assistance on NMR characterization.es_ES
dc.subjectcircular dichroismes_ES
dc.subjectFusion enzymeses_ES
dc.subjectProtein engineeringes_ES
dc.titlePreparation and characterization of a bifunctional aldolase/kinase enzyme. A more efficient biocatalyst for C-C bond formationes_ES
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