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Abstract
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 °C, and it was stable at pH 4.0-11.0 below 40 °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 °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.