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

• Published in: Biochimica et biophysica acta Vol. 1760; no. 2; pp. 191 - 199
• Main Authors: Rhimi, Moez, Bejar, Samir
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2006; Elsevier
• Subjects: Aldose-Ketose Isomerases - genetics; Aldose-Ketose Isomerases - isolation & purification; Amino Acid Sequence; Base Sequence; Biochemistry, Molecular Biology; Chromatography, High Pressure Liquid; Cloning; Cloning, Molecular; Cobalt - pharmacology; d-tagatose; Enzyme Stability; Geobacillus stearothermophilus - enzymology; Geobacillus stearothermophilus - genetics; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; l-arabinose isomerase; Life Sciences; Manganese - pharmacology; Metallic ion demand; Molecular Sequence Data; Purification; Recombinant Proteins - isolation & purification; aa; SDS-PAGE; l-arabinose isomerase; d-tagatose; B; RT; Purification; Cloning; kDa; L-AI; Metallic ion demand; HPLC
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2005.11.007

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 Co 2+ and 1 mM Mn 2+. The V max values were calculated to be 41.3 U/mg for l-arabinose and 8.9 U/mg for d-galactose. Their catalytic efficiencies ( k cat /K m) 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.