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A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

Lookup NU author(s): Dr Claire Dumon, Professor William Willats

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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).


Abstract

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths.


Publication metadata

Author(s): Vidal-Melgosa S, Pedersen HL, Schückel J, Arnal G, Dumon C, Amby DB, Monrad RN, Westereng B, Willats WG

Publication type: Article

Publication status: Published

Journal: The Journal of Biological Chemistry

Year: 2015

Volume: 290

Issue: 14

Pages: 9020-9036

Print publication date: 03/04/2015

Online publication date: 03/04/2015

Acceptance date: 05/02/2015

Date deposited: 17/02/2017

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology, Inc.

URL: http://dx.doi.org/10.1074/jbc.M114.630673

DOI: 10.1074/jbc.M114.630673


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