Synthesis of complex oligosaccharides by using a mutated (1,3)-beta-D-glucan endohydrolase from barley.

Complex oligosaccharides with newly formed (1,3)-beta-glycosidic linkages were obtained in good to excellent yields when substituted or unsubstituted alpha-laminaribiosyl fluorides, acting as donors, were condensed onto mono- and disaccharide beta-D-hexopyranoside acceptors by using a (1,3)-beta-D-glycosynthase. These linear and branched (1,3)-beta-linked oligosaccharides could prove to be important in a range of medical, pharmaceutical, and agricultural applications. Furthermore, the observation that the (1,3)-beta-D-glucan glycosynthase accommodates (1,3)-, (1,4),- and (1,6)-beta-oligosaccharides in its acceptor subsites suggests novel, yet unexpected physiological roles for the wild type (1,3)-beta-D-glucan endohydrolase from higher plants.

Mutated barley (1,3)-beta-D-glucan endohydrolases synthesize crystalline (1,3)-beta-D-glucans.

Barley (1,3)-beta-D-glucan endohydrolases (EC ), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with alpha-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with alpha-laminaribiosyl fluoride and 4'-nitrophenyl beta-D-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k(cat) K(m)(-1)) of mutants E231G, E231S, and E231A as glycosynthases are 28.