title: NMR-based structural glycomics for high-throughput screening of carbohydrate-active enzyme specificity.
abstract: We report here the development of a straightforward, sensitive, and quantitative NMR-based method for high-throughput characterization of carbohydrate structure and screening of carbohydrate active enzyme (CAZyme) specificity. Automated assays starting from gene library expression to carbohydrate structure determination directly from crude reaction media have been established and successfully used to screen a library of 4032 CAZymes obtained by combinatorial engineering, at a rate of 480 enzyme variants per day. This allowed one to accurately discriminate 303 enzyme variants with altered specificity. The results demonstrate the potential of high-throughput NMR technology in glycomics, to mine artificial and natural enzyme diversity for novel biocatalysts.
authors.forename: Romain
authors.initials: R
authors.lastname: Irague
authors.forename: Stéphane
authors.initials: S
authors.lastname: Massou
authors.forename: Claire
authors.initials: C
authors.lastname: Moulis
authors.forename: Olivier
authors.initials: O
authors.lastname: Saurel
authors.forename: Alain
authors.initials: A
authors.lastname: Milon
authors.forename: Pierre
authors.initials: P
authors.lastname: Monsan
authors.forename: Magali
authors.initials: M
authors.lastname: Remaud-Siméon
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Portais
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Potocki-Véronèse
mesh_terms: D055162:Biocatalysis; D050260:Carbohydrate Metabolism; D057075:Enzyme Assays / Q000379:methods*; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D054794:Glycomics / Q000379:methods*; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D004798:Enzymes; D009844:Oligosaccharides; D016695:Glycosyltransferases; C033837:alternansucrase