A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes
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A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes. / Vidal Melgosa, Silvia; Pedersen, Henriette Lodberg; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel Buchvaldt; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William George Tycho.
In: Journal of Biological Chemistry, Vol. 290, 2015, p. 9020-9036.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes
AU - Vidal Melgosa, Silvia
AU - Pedersen, Henriette Lodberg
AU - Schückel, Julia
AU - Arnal, Grégory
AU - Dumon, Claire
AU - Amby, Daniel Buchvaldt
AU - Monrad, Rune Nygaard
AU - Westereng, Bjørge
AU - Willats, William George Tycho
N1 - Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
U2 - 10.1074/jbc.M114.630673
DO - 10.1074/jbc.M114.630673
M3 - Journal article
C2 - 25657012
VL - 290
SP - 9020
EP - 9036
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
ER -
ID: 131740828