Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester
Artikel i vetenskaplig tidskrift, 2015

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.

benzyl

enzymatic assay

Michaelis-Menten parameter

glucuronoyl esterase

enzyme kinetics

estimation

glucuronic acid

glucuronic acid ester

Författare

Hampus Sunner

Chalmers, Biologi och bioteknik, Industriell bioteknik

M. D. Charavgi

National Technical University of Athens

Lisbeth Olsson

Chalmers, Biologi och bioteknik, Industriell bioteknik

Wallenberg Wood Science Center (WWSC)

E. Topakas

National Technical University of Athens

P. Christakopoulos

Luleå tekniska universitet

Molecules

1420-3049 (ISSN)

Vol. 20 17807-17817

Ämneskategorier

Organisk kemi

DOI

10.3390/molecules201017807