title: All-in-one assay for β-d-galactoside sialyltransferases: Quantification of productive turnover, error hydrolysis, and site selectivity.
abstract: Sialyltransferases are important enzymes of glycobiology and the related biotechnologies. The development of sialyltransferases calls for access to quick, inexpensive, and robust analytical tools. We have established an assay for simultaneous characterization of sialyltransferase activity, error hydrolysis, and site selectivity. The described assay does not require expensive substrates, is very sensitive (limit of detection=0.3 μU), and is easy to perform. It is based on sialylation of nitrophenyl galactosides; the products thereof are separated and quantified by ion pair reversed phase high-performance liquid chromatography with ultraviolet detection.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, 8010 Graz, Austria.
authors.forename: Tibor
authors.initials: T
authors.lastname: Czabany
authors.affiliation: Austrian Centre of Industrial Biotechnology, 8010 Graz, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schmölzer
authors.affiliation: Austrian Centre of Industrial Biotechnology, 8010 Graz, Austria.
authors.forename: Christiane
authors.initials: C
authors.lastname: Luley-Goedl
authors.affiliation: Austrian Centre of Industrial Biotechnology, 8010 Graz, Austria.
authors.forename: Doris
authors.initials: D
authors.lastname: Ribitsch
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, 8010 Graz, Austria; Austrian Centre of Industrial Biotechnology, 8010 Graz, Austria. Electronic address: bernd.nidetzky@tugraz.at.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D055162:Biocatalysis; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D057075:Enzyme Assays / Q000379:methods*; D005697:Galactosides / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D005697:Galactosides; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid
keywords: Assay; Donor hydrolysis; Ion pair chromatography; Sialyltransferase; Site selectivity