Engineering L-arabinose isomerase from Enterococcus faecium

DE SOUSA, MARYLANE; MAMMARELLA, ENRIQUE J.; GARCÍA LÓPEZ, JOSE LUIS; PESSELA JOAO, BENEVIDES C.C.; MANZO, RICARDO M.; ROCHA BARRO GONÇALVES, LUCIANA

MOLECULES

MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL-MDPI

Lugar: Basel; Año: 2017 vol. 22 p. 1 - 13

1420-3049

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 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.