Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis
Abstractl-Arabinose isomerase (EC 22.214.171.124) (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. View Full-Text
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de Sousa, M.; Manzo, R.M.; García, J.L.; Mammarella, E.J.; Gonçalves, L.R.B.; Pessela, B.C. Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis. Molecules 2017, 22, 2164.
de Sousa M, Manzo RM, García JL, Mammarella EJ, Gonçalves LRB, Pessela BC. Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis. Molecules. 2017; 22(12):2164.Chicago/Turabian Style
de Sousa, Marylane; Manzo, Ricardo M.; García, José L.; Mammarella, Enrique J.; Gonçalves, Luciana R.B.; Pessela, Benevides C. 2017. "Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis." Molecules 22, no. 12: 2164.
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