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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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Autores principales: Sunner, Hampus, Charavgi, Maria-Despoina, Olsson, Lisbeth, Topakas, Evangelos, Christakopoulos, Paul
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332307/
https://www.ncbi.nlm.nih.gov/pubmed/26404219
http://dx.doi.org/10.3390/molecules201017807
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author Sunner, Hampus
Charavgi, Maria-Despoina
Olsson, Lisbeth
Topakas, Evangelos
Christakopoulos, Paul
author_facet Sunner, Hampus
Charavgi, Maria-Despoina
Olsson, Lisbeth
Topakas, Evangelos
Christakopoulos, Paul
author_sort Sunner, Hampus
collection PubMed
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.
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spelling pubmed-63323072019-01-24 Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester Sunner, Hampus Charavgi, Maria-Despoina Olsson, Lisbeth Topakas, Evangelos Christakopoulos, Paul Molecules Article 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. MDPI 2015-09-25 /pmc/articles/PMC6332307/ /pubmed/26404219 http://dx.doi.org/10.3390/molecules201017807 Text en © 2015 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sunner, Hampus
Charavgi, Maria-Despoina
Olsson, Lisbeth
Topakas, Evangelos
Christakopoulos, Paul
Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester
title 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_short 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332307/
https://www.ncbi.nlm.nih.gov/pubmed/26404219
http://dx.doi.org/10.3390/molecules201017807
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