Directed evolution of new glycosynthases from Agrobacterium β-glucosidase: a general screen to detect enzymes for oligosaccharide synthesis

AbstractBackground: Oligosaccharide synthesis is becoming increasingly important to industry as diverse therapeutic roles for these molecules are discovered. The chemical synthesis of oligosaccharides on an industrial scale is often prohibitively complex and costly. An alternative, that of enzymatic synthesis, is limited by the difficulty of obtaining an appropriate enzyme. A general screen for enzymes that catalyze the synthesis of the glycosidic bond would enable the identification and engineering of new or improved enzymes.Results: Glycosynthases are nucleophile mutants of retaining glycosidases that efficiently catalyze the synthesis of the glycosidic linkage by condensing an activated glycosyl fluoride donor with a suitable acceptor sugar. A novel agar plate-based coupled-enzyme screen was developed (using a two-plasmid system) and used to select an improved glycosynthase from a library of mutants.Conclusions: Plate-based coupled-enzyme screens of this type are extremely valuable for identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases