Artículo
Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
de Souza, Marylane; Manzo, Ricardo Martín
; Garcia, Jose Luis; Mammarella, Enrique José
; Gonçalves, Luciana; Pessela, Benevides
![Icon](/themes/CONICETDigital/images/conicet.png)
![Icon](/themes/CONICETDigital/images/conicet.png)
Fecha de publicación:
12/2017
Editorial:
Molecular Diversity Preservation International
Revista:
Molecules
ISSN:
1420-3049
Idioma:
Inglés
Tipo de recurso:
Artículo publicado
Clasificación temática:
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.
Palabras clave:
L-Arabinose Isomerase
,
Recombinant Dna
,
Affinity Purification
,
D-Tagatose
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Articulos(INTEC)
Articulos de INST.DE DES.TECNOL.PARA LA IND.QUIMICA (I)
Articulos de INST.DE DES.TECNOL.PARA LA IND.QUIMICA (I)
Citación
Garcia, Jose Luis; Mammarella, Enrique José; Gonçalves, Luciana; Manzo, Ricardo Martín; de Souza, Marylane; Pessela, Benevides; et al.; Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis; Molecular Diversity Preservation International; Molecules; 22; 12; 12-2017; 2164
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