Purification and characterization of a thermostable α-galactosidase from Thermoanaerobacterium polysaccharolyticum

Food ingredients containing α-1,6-galactoside bonds elicit gastrointestinal disturbances in monogastric animals, including humans. Pretreatment of such ingredients with α-galactosidase (EC 3.2.1.22) has the potential to alleviate this condition. For this purpose, a thermostable α-galactosidase from Thermoanaerobacterium polysaccharolyticum was purified by a combination of anion exchange and size exclusion chromatographies. The enzyme has a monomeric molecular weight of ∼80 kDa; however, it is active as a dimer. The optimum temperature for enzyme activity is 77.5 °C. Approximately 84 and 88% of enzyme activity remained after 36.5 h of incubation at 70 and 65 °C, respectively. Optimum activity was observed at pH 8.0, with a broad range of activity from pH 5.0 to 9.0. Different transition metals had weak to strong inhibitory effects on enzyme activity. The K_m and V_max of the enzyme are 0.29-0.345 mM and 200-232 μmol/min/mg of protein, respectively. Importantly, enzyme activity was only slightly inhibited by 75-100 mM galactose, an end product of hydrolysis. Enzyme activity was specific for the α-1,6-galactosyl bond, and activity was demonstrated on melibiose and soy molasses.