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Molecules 2017, 22(12), 2164; doi:10.3390/molecules22122164

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

1
Department of Chemical Engineering, Federal University of Ceará, Campus do Pici, BL 709, Fortaleza-CE 60455-760, Brazil
2
Food and Biotechnology Engineering Group, Institute of Technological Development for the Chemical Industry, National University of the Litoral (UNL), National Council of Scientific and Technical Research (CONICET), RN 168 Km 472 “Paraje El Pozo” S/N, S3000 Santa Fe, Argentina
3
Center for Biological Research, CIB, Higher Council for Scientific Research, CSIC, C/Ramiro de Maeztu, 9, 28040 Madrid, Spain
4
Department of Food Biotechnology and Microbiology, Institute of Research in Food Sciences, CIAL, Higher Council for Scientific Research, CSIC, C/Nicolás Cabrera 9, UAM Campus, 28049 Madrid, Spain
5
Department of Engineering and Technology, Polytechnic Institute of Sciences and Technology, Av. Luanda Sul, Rua Lateral Via S10, P.O. Box 1316, Talatona-Luanda Sul, Angola
*
Authors to whom correspondence should be addressed.
Received: 11 August 2017 / Revised: 14 November 2017 / Accepted: 29 November 2017 / Published: 6 December 2017
(This article belongs to the Section Natural Products)
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Abstract

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. View Full-Text
Keywords: l-arabinose isomerase; recombinant DNA; affinity purification; d-tagatose; d-galactose l-arabinose isomerase; recombinant DNA; affinity purification; d-tagatose; d-galactose
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MDPI and ACS Style

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.

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