Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester

Molecules

Hampus Sunner¹  Maria-Despoina Charavgi²  Lisbeth Olsson¹  Evangelos Topakas²  Paul Christakopoulos³ 

[1] Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg SE-412 96, Sweden; E-Mails:;Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece; E-Mails:;Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, Luleå SE-971 87, Sweden

关键词: glucuronic acid;    glucuronoyl esterase;    enzymatic assay;    benzyl glucuronic acid ester;    enzyme kinetics;    Michaelis-Menten parameter estimation;   

DOI  :  10.3390/molecules201017807

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.