Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive L-arabinose isomerase from the Bacillus stearothermophilus US100 strain Moez Rhimi, Samir Bejar ⁎ Laboratoire d'Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax BP “K” 3038 Sfax, Tunisie Received 17 May 2005; received in revised form 9 November 2005; accepted 9 November 2005 Available online 13 December 2005 Abstract 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. © 2005 Elsevier B.V. All rights reserved. Keywords: L-arabinose isomerase; Cloning; Purification; Metallic ion demand; D-tagatose 1. Introduction The enzyme L-arabinose isomerase permits the conversion of L-arabinose to L-ribulose inside biological systems. It is also referred to as D-galactose isomerase due to its ability to isomerize the D-galactose into D-tagatose. This later ketohexose is certainly of importance, when one considers the privileged position of this isomer of D-galactose within sweeteners. It is a rare natural sugar having a taste and physical properties similar to sucrose [1]. In addition, D-tagatose is an anti-hyperglyce- miant factor with a very low calorie carbohydrate and bulking agent [2–4]. D-tagatose is also an efficient anti-biofilm which can be used as a cytoprotective supplement for the storage of organs to reduce the reperfusion injury [5,6]. It was the subject of recent interests in food and drug industry and was considered as a safe and low calorie substrate in the United States [7]. However, the use of this sugar is limited due to its high cost. Recently, a biological mean of D-tagatose production has been developed as an easy feasible and environmentally clean procedure. Many micro-organisms such as Arthrobacter, Lactobacillus, Mycobacterium, klebsiella and Gluconobacter were recognized for their ability to convert D-galactitol into D-tagatose [8–11]. However, the D-galactitol is an expensive substrate which appears to have a low potential for industrial application. In order to overcome this limit, Cheetam and Wootton [12] proposed a new bioprocess to convert D-galactose into D- tagatose via the L-arabinose isomerase of Lactobacillus and Mycobacterium. In this context, it is worth noting that the rate of D-galactose bioconversion into D-tagatose had a close Biochimica et Biophysica Acta 1760 (2006) 191 – 199 http://www.elsevier.com/locate/bba Abbreviations: B, Bacillus; L-AI, L-arabinose isomerase; kDa, kilodalton; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; aa, amino acid; RT, retention time ⁎ Corresponding author. Tel.: +216 74 44 04 51; fax: +216 74 44 04 51. E-mail address: Samir.bejar@cbs.rnrt.tn (S. Bejar). 0304-4165/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2005.11.007