Cloning, expression and characterization of l-arabinose isomerase from Thermotoga neapolitana : bioconversion of d-galactose to d-tagatose using the enzyme

Abstract

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 Co 2þ and Mn 2þ 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 Wmol min 31 mg 31) and 250 mM (v max , 14.3 Wmol min 31 mg 31), respectively, that were determined in the presence of both 1 mM Co 2þ and 1 mM Mn 2þ . A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 C. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.