Glycosynthases are engineered retaining glycosidases devoid of hydrolase activity that efficiently catalyze transglycosylation reactions. The mechanism of the glycosynthase reaction is probed with the E134A mutant of Bacillus licheniformis 1,3-1,4-beta-glucanase. This endo-glycosynthase is regiospecific for formation of a beta-1,4-glycosidic bond with alpha-glycosyl fluoride donors (laminaribiosyl as the minimal donor) and oligosaccharide acceptors containing glucose or xylose on the nonreducing end (aryl monosaccharides or oligosaccharides). The pH dependence of the glycosynthase activity reflects general base catalysis with a kinetic pK(a) of 5.2 +/- 0.1. Kinetics of enzyme inactivation by a water-soluble carbodiimide (EDC) are consistent with modification of an active site carboxylate group with a pK(a) of 5.3 +/- 0.2. The general base is Glu138 (the residue acting as the general acid-base in the parental wild-type enzyme) as probed by preparing the double mutant E134A/E138A. It is devoid of glycosynthase activity, but use of sodium azide as an acceptor not requiring general base catalysis yielded a beta-glycosyl azide product. The pK(a) of Glu138 (kinetic pK(a) on k(cat)/K(M) and pK(a) of EDC inactivation) for the E134A glycosynthase has dropped 1.8 pH units compared to the pK(a) values of the wild type, enabling the same residue to act as a general base in the glycosynthase enzyme. Kinetic parameters of the E134A glycosynthase-catalyzed condensation between Glcbeta4Glcbeta3GlcalphaF (2) as a donor and Glcbeta4Glcbeta-pNP (15) as an acceptor are as follows: k(cat) = 1.7 s(-)(1), K(M)(acceptor) = 11 mM, and K(M)(donor) < 0.3 mM. Donor self-condensation and elongation reactions are kinetically evaluated to establish the conditions for preparative use of the glycosynthase reaction in oligosaccharide synthesis. Yields are 70-90% with aryl monosaccharide and cellobioside acceptors, but 25-55% with laminaribiosides, the lower yields (and lower initial rates) due to competitive inhibition of the beta-1,3-linked disaccharide acceptor for the donor subsites of the enzyme.