Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: Bioconversion of D-galactose to D-tagatose using the enzyme

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56 677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85°C, and required divalent cations such as Co²⁺ and Mn²⁺ for its activity and thermostability. The apparent K_m values of the enzyme for L-arabinose and D-galactose were 116 mM (v_max, 119 μmol min^-1 mg^-1) and 250 mM (v_max, 14.3 μmol min^-1 mg^-1), respectively, that were determined in the presence of both 1 mM Co²⁺ and 1 mM Mn²⁺. A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80°C.