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

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Silvia Vidal Melgosa, Henriette Lodberg Pedersen, Julia Schückel, Grégory Arnal, Claire Dumon, Daniel Buchvaldt Amby, Rune Nygaard Monrad, Bjørge Westereng, William George Tycho Willats

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing, together with associated bioinformatic 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 semi-quantitative enzyme-screening technique which requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme cocktails and crude culture broths against single substrates, substrate mixtures and biomass samples. Moreover, we show that the technique can be used to analyse 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 un-characterised enzymes and by screening enzyme activities from fungal culture broths.

TidsskriftJournal of Biological Chemistry
Sider (fra-til)9020-9036
Antal sider17
StatusUdgivet - 2015

ID: 131740828