title: Screening glycosynthase libraries with a fluoride chemosensor assay independently of enzyme specificity: identification of a transitional hydrolase to synthase mutant.
abstract: Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme-directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. In the present paper, we report a general screening assay that is independent of enzyme specificity, and implemented in an HTS (high-throughput screening) format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept, it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond the expected mutations at the glutamic acid (catalytic) nucleophile, other variants have been shown to acquire glycosynthase activity. Surprisingly, an aspartic acid for glutamic acid replacement renders a highly active glycosynthase, but still retains low hydrolase activity. It appears as an intermediate state between glycosyl hydrolase and glycosynthase.
authors.affiliation: *Laboratory of Biochemistry, Institut Químic de Sarria, Universitat Ramon Llull, 08017 Barcelona, Spain.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Andrés
authors.affiliation: *Laboratory of Biochemistry, Institut Químic de Sarria, Universitat Ramon Llull, 08017 Barcelona, Spain.
authors.forename: Hugo
authors.initials: H
authors.lastname: Aragunde
authors.affiliation: *Laboratory of Biochemistry, Institut Químic de Sarria, Universitat Ramon Llull, 08017 Barcelona, Spain.
authors.forename: Antoni
authors.initials: A
authors.lastname: Planas
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005459:Fluorides / Q000737:chemistry*; D006006:Glycogen Synthase / Q000737:chemistry / Q000235:genetics*; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D054852:Small Molecule Libraries / Q000737:chemistry*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D054852:Small Molecule Libraries; D006006:Glycogen Synthase; D006867:Hydrolases; D005459:Fluorides