Biochemical properties of L-arabinose isomerase from Clostridium hylemonae to produce D-tagatose as a functional sweetener

d -Tagatose has gained substantial interest due to its potential functionalities as a sucrose substitute. In this study, the gene araA , encoding l -arabinose isomerase ( l -AI) from Clostridium hylemonae (DSM 15053), was cloned and expressed in Escherichia coli BL21 (DE3). This gene consists of 1,506 nucleotides and encodes a protein of 501 amino acid residues with a calculated molecular mass of 56,554 Da. Since l -AI was expressed as an intracellular inclusion body, this enzyme was solubilized with guanidine hydrochloride, refolded, and activated with a descending concentration gradient of urea. The purified enzyme exhibited the greatest activity at 50°C, pH 7–7.5, and required 1 mM of Mg 2+ as a cofactor. Notably, the catalytic efficiency (3.69 mM -1 sec -1 ) of l -AI from C . hylemonae on galactose was significantly greater than that of other previously reported enzymes. The bioconversion yield of d -tagatose using the C . hylemonae l -arabinose isomerase at 60°C reached approximately 46% from 10 mM of d -galactose after 2 h. From these results, it is suggested that the l -arabinose isomerase from C . hylemonae could be utilized as a potential enzyme for d -tagatose production due to its high conversion yield at an industrially competitive temperature.