title: Development of a colourimetric assay for glycosynthases.
abstract: The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration. In the present paper, we describe a new version of a glycosynthase activity assay using a silyl ether of p-nitrophenol, allowing fast reliable detection of fluoride even at concentrations of 4mM and higher. This improvement of detection allows not only screening and identification but also kinetic characterisation of glycosynthases and synthetic reactions in a fast microtiter plate format. The applicability of the assay was successfully demonstrated by the biochemical characterisation of the mesophilic β-glucosynthase of Abg-E358S (Rhizobium radiobacter) and psychrotolerant β-glucosynthase BglU-E377A (Micrococcus antarcticus). The limitation of hyperthermophilic glycosidases as potential glycosynthases, when using glycosyl fluoride donors, was also illustrated by the example of the putative β-galactosidase GalPf from Pyrococcus furiosus.
authors.affiliation: Heinrich-Heine-Universität Düsseldorf, Institut für Bioorganische Chemie im Forschungszentrum Jülich, Gebäude: 15.8, 52426 Jülich, Germany. Electronic address: m.hayes@fz-juelich.de.
authors.forename: Marc R
authors.initials: MR
authors.lastname: Hayes
authors.affiliation: Forschungszentrum Jülich, IBG-1: Biotechnology, 52425 Jülich, Germany. Electronic address: k.bochinsky@fz-juelich.de.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Bochinsky
authors.affiliation: Heinrich-Heine-Universität Düsseldorf, Institut für Bioorganische Chemie im Forschungszentrum Jülich, Gebäude: 15.8, 52426 Jülich, Germany. Electronic address: lisa.seibt@uni-duesseldorf.de.
authors.forename: Lisa S
authors.initials: LS
authors.lastname: Seibt
authors.affiliation: RWTH Aachen University, Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz Institute for Biomedical Engineering, Sammelbau Biologie, Worringer Weg 1, D-52056 Aachen, Germany. Electronic address: l.elling@biotec.rwth-aachen.de.
authors.forename: Lothar
authors.initials: L
authors.lastname: Elling
authors.affiliation: Heinrich-Heine-Universität Düsseldorf, Institut für Bioorganische Chemie im Forschungszentrum Jülich, Gebäude: 15.8, 52426 Jülich, Germany; Forschungszentrum Jülich, IBG-1: Biotechnology, 52425 Jülich, Germany. Electronic address: j.pietruszka@fz-juelich.de.
authors.forename: Jörg
authors.initials: J
authors.lastname: Pietruszka
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002384:Catalysis; D003124:Colorimetry / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005459:Fluorides / Q000032:analysis; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000032:analysis* / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D007700:Kinetics; D009596:Nitrophenols; D017354:Point Mutation; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D006027:Glycosides; D009596:Nitrophenols; D006026:Glycoside Hydrolases; D005459:Fluorides; C024836:4-nitrophenol
keywords: Chemosensor; Enzyme characterisation; Fluoride detection; Glycosynthase; Screening