Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. They are engineered retaining glycosidases in which the hydrolase activity has been abolished by mutation of the catalytic nucleophile but efficiently catalyse transglycosylation when using activated glycosyl fluoride donors with the opposite anomeric configuration to the substrate in the wild-type enzyme. The need of new specificities (i.e non-natural substrates), applications, and performance improvements, is being addressed by enzyme directed evolution. These strategies largely depend on high-throughput screening methods. Most reported methodologies focus on the detection of the product formed in the glycosynthase reaction, thus being enzyme-dependent.(1) Only one “universal” screening method has been proposed, a pH-based assay that measures the hydrofluoric acid released as by-product of the glycosynthase reaction, being detected by color change of a pH-indicator.(2) However, it is not very reproducible and difficult to implement due to matrix sample variations.
We have developed a new general screening assay independent of enzyme specificity for the screening of glycosynthases libraries.(3) This assay is based on the use of a chemical sensor to transduce fluoride concentration (by-product of all glycosynthase reactions using fluoride activated donors) into a fluorescence signal. We report here the application to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond to the expected mutations at the Glu (catalytic) nucleophile, other variants have shown to acquire glycosynthase activity. Surprisingly, the Asp mutation renders a highly active glycosynthase. This variant has been characterized in detail and provides mechanistic insights on the role of neighbouring residues in the glycosynthase mechanism.
1. Roman K., Withers S.G. (2010) Carbohydr. Res. 345, 1272-1279 15, 546-551
2. Ben-Davis A., Shoham G., Shoham Y.. (2008) Chem. Biol.
3. Andrés E., Aragunde H., Planas A., Screening directed evolution libraries for glycosynthase activity independent of enzyme specificity with a fluorescent fluoride sensor. Submitted.
