Towards efficient enzymatic conversion of D-galactose to D-tagatose: purification and characterization of L-arabinose isomerase from Lactobacillus brevis.

L-arabinose isomerase (L-AI) (EC 5. 3. 1. 4. L-AI) that mediates the isomerization of D-galactose to D-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1 kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The L-AI was found to be stable at 45-75 °C, and at pH 7.0-9.0. Its optimum temperature and pH was determined as 65 °C and 7.0, respectively. Besides, we found that Ca ²⁺ , Cu ²⁺ , and Ba ²⁺ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg ²⁺ , Mn ²⁺ , or Co ²⁺ ions. The optimum concentration of Mn ²⁺ and Co ²⁺ was determined to be 1 mM. Furthermore, we characterized the kinetic parameters for L-AI and determined the K _m (129 mM) and the V _max (0.045 mM min ^- 1 ) values. Notably, L. brevisL-AI exhibited a high bioconversion yield of 43% from D-galactose to D-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other L-AIs from various organisms.