Glycosynthase activity of Geobacillus stearothermophilus GH52 β-xylosidase: Efficient synthesis of xylooligosaccharides from α-D-xylopyranosyl fluoride through a conjugated reaction

Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 β-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using α-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction of α-D-xylopyranosyl fluoride, providing mainly α-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis-Menten behavior, with kinetic constants of 1.3 s^-1 and 2.2 mM for k_cat and K_M(acceptor) respectively, and a k_cat/ KM(acceptor) value of 0.59 s^-1 mM^-1. When the β-xylqsldase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable α-D-xylopyranosyl fluoride as the sole substrate.