Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive l-arabinose isomerase from the Bacillus stearothermophilus US100 strain

The araA gene encoding l-arabinose isomerase from Bacillus stearothermophilus US100 strain was cloned, sequenced and over-expressed in E. coli. This gene encodes a 496-amino acid protein with a calculated molecular weight of 56.161 kDa. Its amino acid sequence displays the highest identity with L-AI from Thermus sp. IM6501 (98%) and that of Geobacillus stearothermophilus T6 (97%). According to SDS-PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of nearly 225 kDa, composed of four identical 56-kDa subunits. The L-AI US100 was optimally active at pH 7.5 and 80 °C. It was distinguishable by its behavior towards divalent ions. Indeed, the L-AI US100 activity and thermostability were totally independent for metallic ions until 65 °C. At temperatures above 65 °C, the enzyme was also independent for metallic ions for its activity but its thermostability was obviously improved in presence of only 0.2 mM Co2+ and 1 mM Mn2+. The Vmax values were calculated to be 41.3 U/mg for l-arabinose and 8.9 U/mg for d-galactose. Their catalytic efficiencies (kcat/Km) for l-arabinose and d-galactose were, respectively, 71.4 and 8.46 mM−1 min−1. L-AI US100 converted the d-galactose into d-tagatose with a high conversion rate of 48% after 7 h at 70 °C.