INVESTIGADORES
MAMMARELLA Enrique Jose
artículos
Título:
Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
Autor/es:
DE SOUZA, MARYLANE; MANZO, RICARDO M.; GARCIA, JOSÉ; MAMMARELLA, ENRIQUE J.; GONÇALVES, LUCIANA; PESSELA, BENEVIDES
Revista:
MOLECULES
Editorial:
MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL-MDPI
Referencias:
Lugar: Basel; Año: 2017
ISSN:
1420-3049
Resumen:
L-Arabinose isomerase (EC 5.3.1.4) (L-AI) from Enterococcus faecium DBFIQ E36 wasoverproduced 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 theN-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, 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.