Globotriose (Gal alpha 1-4Gal beta 1-4Glc) is an important cell surface epitope that acts as the receptor for Shiga-like toxins, and it is also the core structure of Globo H and SSEA4 that are tumor-associated glycans. Hence, the enzymatic synthesis of globotriose would be necessary for the development of carbohydrate-based therapeutics for bacterial infections and cancers. Here, a novel GH27 alpha-galactosidase gene (agaBf3S), a 1521-bp DNA encoding 506 amino acids with a calculated molecular mass of 57.7 kDa, from Bacteroides fragilis NCTC9343 was cloned and heterogeneously expressed in Escherichia coli. The recombinant enzyme AgaBf3S preferentially hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (pNP alpha Gal) in all tested nitrophenyl glycosides. It showed maximum activity at pH 4.5 and 40 degrees C, and it was stable at pH 4.0-11.0 below 40 degrees C and metal-independent. The K-m and k(cat) values for pNP alpha Gal, melibiose, and globotriose were 1.27 mM and 172.97 S-1, 62.76 mM and 17.74 S-1, and 4.62 mM and 388.45 S-1, respectively. AgaBf3S could transfer galactosyl residue from pNP alpha Gal to lactose (Gal beta 1-4Glc) with high efficiency and strict alpha 1-4 regioselectivity. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation reaction catalyzed by AgaBf3S were studied in detail. AgaBf3S could synthesize globotriose as a single transglycosylation product with a maximum yield of 32.4 % from 20 mM pNP alpha Gal and 500 mM lactose (pH 4.5) at 40 degrees C for 30 min. This new one-enzyme one-step synthetic reaction is simple, fast, and low cost, which provides a promising alternative to the current synthetic methods for access to pharmaceutically important Gal alpha 1-4-linked oligosaccharides.