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

Abstract

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 50C, pH 7–7.5, and required 1 mM of Mg2+ as a cofactor. Notably, the catalytic efficiency (3.69 mM-1sec-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 60C 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.