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

Hampus Sunner (Institutionen för biologi och bioteknik, Industriell bioteknik) ; M. D. Charavgi ; Lisbeth Olsson (Institutionen för biologi och bioteknik, Industriell bioteknik) ; E. Topakas ; P. Christakopoulos
Molecules (1420-3049). Vol. 20 (2015), 10, p. 17807-17817.
[Artikel, refereegranskad vetenskaplig]

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.

Nyckelord: glucuronic acid, glucuronoyl esterase, enzymatic assay, benzyl, glucuronic acid ester, enzyme kinetics, Michaelis-Menten parameter, estimation



Denna post skapades 2016-01-07. Senast ändrad 2016-05-24.
CPL Pubid: 230051

 

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Institutioner (Chalmers)

Institutionen för biologi och bioteknik, Industriell bioteknik

Ämnesområden

Organisk kemi

Chalmers infrastruktur