The nonnucleophilic mutant E383A β-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of α-glucosyl and α-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 °C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pKa of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, β-(1→3) disaccharides are obtained in good to excellent yields, thus expanding the synthetic products available with current exo-glycosynthases. With xylopyranosyl acceptor, regioselectivity is poorer and results in the formation of a mixture of β-(1→3) and β-(1→4) linkages. In contrast, disaccharide acceptors produce exclusively β-(1→4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from β-(1→3) to β-(1→4). To improve operational performance, the E383A mutant was immobilized on a Ni2+-chelating Sepharose resin. Immobilization did not increase stability to pH and organic solvents, but the operational stability and storage stability were clearly enhanced for recycling and scaling-up.