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

Lisbeth Olsson,Evangelos Topakas,Hampus Sunner,Paul Christakopoulos,Maria-Despoina Charavgi

doi:10.3390/molecules201017807

Abstract

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.

Highlights

Glucuronoyl esterases (GEs) are a recently discovered group of enzymes, which have been attributed the functional role of hydrolyzing the ester bonds between lignin alcohols and the 4-O-methyl-D-glucuronic acid side chains of xylan in plant cell walls [1,2]
As a method for qualitative detection of glucuronoyl esterases (GEs) activity, the TLC assay is a straightforward application of the N-(1-naphthyl)ethylenediamine dihydrochloride-based method [1,17], with the option to use an
The TLC assay methodology was validated on purified PaGE1 and concentrated StGE2 culture filtrate

Summary

Introduction

Glucuronoyl esterases (GEs) are a recently discovered group of enzymes, which have been attributed the functional role of hydrolyzing the ester bonds between lignin alcohols and the 4-O-methyl-D-glucuronic acid side chains of xylan in plant cell walls [1,2]. These ester bonds constitute one of several bond types postulated to link lignin to carbohydrates (LC-bonds) in lignified plant matter [3]. The ScGE1 sequence initiated the establishment of carbohydrate esterase (CE) family 15 in the CAZy classification [6].

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