Subcloning, expression, and molecular characterization of L-arabinose isomerase from Thermotoga thermarum.

D-tagatose is an isomer of L-fructose and around 92% as sweet as sugar. Therefore, it can be used as a low-calorie sweetener that demonstrates an anti-hyperglycemic effect. D-tagatose can be produced through enzymatic conversion of D-galactose by the enzyme L-arabinose isomerase (L-AIs; EC 5.3.1.4). Hence, we describe the expression of L-AI from the hyper-thermophilic bacterium Thermotoga thermarum in Escherichia coli. The araA gene encoding the L-AI from T. thermarum (L-TTAI) was subcloned into the pRHA-vector for the expression in E. coli NiCo21 under the control of a rhamnose-tightly regulated promoter. The His6-tagged enzyme was successfully expressed after the induction with L-rhamnose and subsequently purified using immobilized metal-chelate affinity chromatography (IMAC) on TALON™-matrix. The SDS-PAGE result revealed that the enzyme has a molecular weight of approximately 56 kDa, corresponding to the molecular weight calculated based on its amino acid sequence. The molecular characteristics of the L-TTAI were compared to other bacterial L-arabinose isomerases. [ABSTRACT FROM AUTHOR]