Purification and characterization of an l-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing d-tagatose

Abstract

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 °C and pH 8.5 in the presence of Mn 2+. The enzyme was stable at temperatures below 60 °C and within the pH range 7.5–8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a K i of 5.5 mM. The apparent K m and V max for l-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency ( k cat/ K m) was 48 mM −1 min −1 for l-arabinose and 0.5 mM −1 min −1 for d-galactose. Mn 2+ was a competitive activator and increased the thermal stability of the AI. The d-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn 2+ and 48% with Mn 2+ after 300 min at 65 °C.