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

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from E36 was overproduced in by designing a codon-optimized synthetic 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 -His-l-AI and -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 -His-l-AI was preferentially hexameric in solution, whereas -His-l-AI was mainly monomeric. The specific activity of the -His-l-AI at acidic pH was higher than that of -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, -His-l-AI was more active and stable at alkaline pH than -His-l-AI. -His-l-AI follows a Michaelis-Menten kinetic, whereas -His-l-AI fitted to a sigmoidal saturation curve.