Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A β-glucosidase

The normucleophilic mutant E383A beta-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of alpha-glucosyl and alpha-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 degrees C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pK(a) of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, beta-(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 beta-(1 -> 3) and beta-(1 -> 4) linkages. In contrast, disaccharide acceptors produce exclusively beta-(1 -> 4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from beta-(1 -> 3) to beta-(1 -> 4). To improve operational performance, the E383A mutant was immobilized on a Ni(2+)-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. (c) 2006 Elsevier Ltd. All rights reserved.