title: Comparison of broad-scope assays of nucleotide sugar-dependent glycosyltransferases.
abstract: Glycosyltransferases (GTs) are abundant in nature and diverse in their range of substrates. Application of GTs is, however, often complicated by their narrow substrate specificity. GTs with tailored specificities are highly demanded for targeted glycosylation reactions. Engineering of such GTs is, however, restricted by lack of practical and broad-scope assays currently available. Here we present an improvement of an inexpensive and simple assay that relies on the enzymatic detection of inorganic phosphate cleaved from nucleoside phosphate products released in GT reactions. This phosphatase-coupled assay (PCA) is compared with other GT assays: a pH shift assay and a commercially available immunoassay in Escherichia coli cell-free extract (CE). Furthermore, we probe PCA with three GTs with different specificities. Our results demonstrate that PCA is a versatile and apparently general GT assay with a detection limit as low as 1 mU. The detection limit of the pH shift assay is roughly 4 times higher. The immunoassay, by contrast, detected only nucleoside diphosphates (NDPs) but had the lowest detection limit. Compared with these assays, PCA showed superior robustness and, therefore, appears to be a suitable general screening assay for nucleotide sugar-dependent GTs.
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, A-8010 Graz, Austria.
authors: Patricia
authors: P
authors: Bubner
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, A-8010 Graz, Austria.
authors: Tibor
authors: T
authors: Czabany
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, A-8010 Graz, Austria; Austrian Centre of Industrial Biotechnology, A-8010 Graz, Austria.
authors: Christiane
authors: C
authors: Luley-Goedl
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, A-8010 Graz, Austria; Austrian Centre of Industrial Biotechnology, A-8010 Graz, Austria. Electronic address: bernd.nidetzky@tugraz.at.
authors: Bernd
authors: B
authors: Nidetzky
mesh_terms: D000327:Adsorption; D000469:Alkaline Phosphatase / Q000378:metabolism*; D000537:Aluminum Oxide / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System / Q000201:enzymology / Q000378:metabolism; D002498:Centrifugation; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D057230:Limit of Detection; D009705:Nucleosides / Q000378:metabolism*; D010710:Phosphates / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007202:Indicators and Reagents; D009705:Nucleosides; D010710:Phosphates; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014533:Uridine Diphosphate Glucose Dehydrogenase; D016695:Glycosyltransferases; D012799:Sialyltransferases; C102599:sucrose synthase kinase; D017346:Protein Serine-Threonine Kinases; D000469:Alkaline Phosphatase; D000537:Aluminum Oxide
keywords: Activity assay; Glycosyltransferase; Nucleotide sugar; Phosphatase; Phosphate