title: Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester.
abstract: Research on glucuronoyl esterases (GEs) has been hampered by the lack of enzyme assays based on easily obtainable substrates. While benzyl d-glucuronic acid ester (BnGlcA) is a commercially available substrate that can be used for GE assays, several considerations regarding substrate instability, limited solubility and low apparent affinities should be made. In this work we discuss the factors that are important when using BnGlcA for assaying GE activity and show how these can be applied when designing BnGlcA-based GE assays for different applications: a thin-layer chromatography assay for qualitative activity detection, a coupled-enzyme spectrophotometric assay that can be used for high-throughput screening or general activity determinations and a HPLC-based detection method allowing kinetic determinations. The three-level experimental procedure not merely facilitates routine, fast and simple biochemical characterizations but it can also give rise to the discovery of different GEs through an extensive screening of heterologous Genomic and Metagenomic expression libraries.
authors.affiliation: Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg SE-412 96, Sweden. hampus.sunner@chalmers.se.|Wallenberg Wood Science Centre, Teknikringen 56-58, Stockholm SE-100 44, Sweden. hampus.sunner@chalmers.se.
authors.forename: Hampus
authors.initials: H
authors.lastname: Sunner
authors.affiliation: Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece. chem.marianna@gmail.com.
authors.forename: Maria-Despoina
authors.initials: MD
authors.lastname: Charavgi
authors.affiliation: Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg SE-412 96, Sweden. lisbeth.olsson@chalmers.se.|Wallenberg Wood Science Centre, Teknikringen 56-58, Stockholm SE-100 44, Sweden. lisbeth.olsson@chalmers.se.
authors.forename: Lisbeth
authors.initials: L
authors.lastname: Olsson
authors.affiliation: Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece. vtopakas@chemeng.ntua.gr.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Topakas
authors.affiliation: Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, Luleå SE-971 87, Sweden. paul.christakopoulos@ltu.se.
authors.forename: Paul
authors.initials: P
authors.lastname: Christakopoulos
mesh_terms: D004950:Esterases / Q000737:chemistry*; D004952:Esters; D020723:Glucuronic Acid / Q000737:chemistry*; D007700:Kinetics; D013379:Substrate Specificity
chemical_list: D004952:Esters; D020723:Glucuronic Acid; D004950:Esterases
keywords: Michaelis-Menten parameter estimation; benzyl glucuronic acid ester; enzymatic assay; enzyme kinetics; glucuronic acid; glucuronoyl esterase