Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester
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, li...
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doaj-506da387480a448f9ab3d2d847b7ac232020-11-24T22:23:18ZengMDPI AGMolecules1420-30492015-09-012010178071781710.3390/molecules201017807molecules201017807Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid EsterHampus Sunner0Maria-Despoina Charavgi1Lisbeth Olsson2Evangelos Topakas3Paul Christakopoulos4Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg SE-412 96, SwedenBiotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, GreeceIndustrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg SE-412 96, SwedenBiotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, GreeceBiochemical Process Engineering, Division of Chemical Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, Luleå SE-971 87, SwedenResearch 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.http://www.mdpi.com/1420-3049/20/10/17807glucuronic acidglucuronoyl esteraseenzymatic assaybenzyl glucuronic acid esterenzyme kineticsMichaelis-Menten parameter estimation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Hampus Sunner Maria-Despoina Charavgi Lisbeth Olsson Evangelos Topakas Paul Christakopoulos |
spellingShingle |
Hampus Sunner Maria-Despoina Charavgi Lisbeth Olsson Evangelos Topakas Paul Christakopoulos Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester Molecules glucuronic acid glucuronoyl esterase enzymatic assay benzyl glucuronic acid ester enzyme kinetics Michaelis-Menten parameter estimation |
author_facet |
Hampus Sunner Maria-Despoina Charavgi Lisbeth Olsson Evangelos Topakas Paul Christakopoulos |
author_sort |
Hampus Sunner |
title |
Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester |
title_short |
Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester |
title_full |
Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester |
title_fullStr |
Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester |
title_full_unstemmed |
Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester |
title_sort |
glucuronoyl esterase screening and characterization assays utilizing commercially available benzyl glucuronic acid ester |
publisher |
MDPI AG |
series |
Molecules |
issn |
1420-3049 |
publishDate |
2015-09-01 |
description |
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. |
topic |
glucuronic acid glucuronoyl esterase enzymatic assay benzyl glucuronic acid ester enzyme kinetics Michaelis-Menten parameter estimation |
url |
http://www.mdpi.com/1420-3049/20/10/17807 |
work_keys_str_mv |
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