title: Oligosaccharide biosensor for direct monitoring of enzymatic activities using QCM-D.
abstract: Enzymatic modification of saccharidic biomass is a subject of intensive research with potential applications in plant or human health, design of biomaterials and biofuel production. Bioengineering and metagenomics provide access to libraries of glycoside hydrolases but the biochemical characterization of these enzymes remains challenging, requiring fastidious colorimetric tests in discontinuous assays. Here, we describe a highly sensitive carbohydrate biosensor for the detection and characterization of glycoside hydrolases. Immobilization of oligosaccharides was achieved using copper-catalyzed azide-alkyne cycloaddition of maltoheptaose-modified probes onto self-assembled monolayers bearing azide reactive groups. This biosensor allowed detection of glycoside hydrolase activities at the picomolar level using quartz-crystal microbalance with dissipation monitoring (QCM-D). To our knowledge, this protocol provides the best performance to date for the detection of glycoside hydrolase activities. For each enzyme tested, we could determine the kinetic constant from the QCM-D data, and derive conclusions that correlated well with those of standard colorimetric tests. This opens the way to a new generation of rapid and direct tests characterizing functionally carbohydrate-active enzymes.
authors.affiliation: Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS), affiliated with Université de Grenoble, 38041 Grenoble cedex 9, France. aurelie.bouchet-spinelli@ujf-grenoble.fr
authors.forename: Aurélie
authors.initials: A
authors.lastname: Bouchet-Spinelli
authors.forename: Bertrand
authors.initials: B
authors.lastname: Reuillard
authors.forename: Liliane
authors.initials: L
authors.lastname: Coche-Guérente
authors.forename: Sylvie
authors.initials: S
authors.lastname: Armand
authors.forename: Pierre
authors.initials: P
authors.lastname: Labbé
authors.forename: Sébastien
authors.initials: S
authors.lastname: Fort
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D015374:Biosensing Techniques / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods; D006026:Glycoside Hydrolases / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D058525:Quartz Crystal Microbalance Techniques / Q000379:methods*
chemical_list: D009844:Oligosaccharides; D006026:Glycoside Hydrolases
keywords: Carbohydrate biosensor; Carbohydrate-active enzyme; Oligosaccharides; Quartz-crystal microbalance with dissipation monitoring (QCM-D).; Self-assembled monolayers