Glycoside hydrolases are a widespread group of enzymes hydrolyzing the glycosidic bond between two or more carbohydrates. Catalytic mutants of glycosidases were found to be able to act as glycosynthases. In these enzymes the catalytic nucleophile is replaced by a smaller non-nucleophilic residue, resulting in practically inactive hydrolase. However, in the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes transfer the activated sugars to suitable acceptors (Mackenzie et al. JACS, 1998). GanB is a β-galactosidase isolated from the thermophilic bacterium Geobacillus stearothermophilus T-6. The enzyme belongs to family 42 of glycoside hydrolases (GH42) acting with a retaining mechanism and capable of hydrolyzing short β-1,4 linked galactosaccharides and the synthetic substrate para-nitrophenyl-β-D-galactopyranoside (Tabachnikov & Shoham, FEBS J. 2013). A nucleophile mutant of GanB, GanB-E323A, can function as glycosynthase using α-D-galactopyranosyl- and β-L-arabinopyranosyl fluoride as donors and various aryl-glycosides as acceptors, creating di- and trisaccharide products. The mutant enzyme can also catalyze the self-condensation reaction of α-D-galactopyranosyl fluoride, producing mainly α-D-galactobiosyl fluoride. This work expands the repertoire of known glycosynthases, since GanB is the first member of GH42 family that was successfully converted into glycosynthase, and the obtained oligosaccharide products may be used for various applications in medicine and food industry. Directed evolution approaches are been used to enhance the catalytic power of GanB-E323A and increase the product yield.
