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Título: | Engineering the L-Arabinose somerase from Enterococcus faecium for D-tagatose synthesis |
Autor: | Sousa, Marylane de; Manzo, Ricardo M.; García, José Luis CSIC ORCID ; Mammarella, Enrique J.; Gonçalves, Luciana R. B.; Pessela, Benevides C. CSIC ORCID | Palabras clave: | L-arabinose isomerase Recombinant DNA Affinity purification D-tagatose D-galactose |
Fecha de publicación: | 2017 | Editor: | Multidisciplinary Digital Publishing Institute | Citación: | Molecules 22(12): 2164 (2017) | Resumen: | L-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-L-AI was preferentially hexameric in solution, whereas N-His-L-AI was mainly monomeric. The specific activity of the N-His-L-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-L-AI was more active and stable at alkaline pH than N-His-L-AI. N-His-L-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve. | Versión del editor: | https://doi.org/10.3390/molecules22122164 | URI: | http://hdl.handle.net/10261/194069 | DOI: | 10.3390/molecules22122164 | ISSN: | 1420-3049 |
Aparece en las colecciones: | (CIAL) Artículos (CIB) Artículos |
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enginsynthe.pdf | 1,82 MB | Adobe PDF | Visualizar/Abrir |
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