TY - JOUR

T1 - A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes

AU - Kracun, Stjepan Kresimir

AU - Schückel, Julia

AU - Westereng, Bjørge

AU - Thygesen, Lisbeth Garbrecht

AU - Monrad, Rune Nygaard

AU - Eijsink, Vincent G. H.

AU - Willats, William George Tycho

PY - 2015

Y1 - 2015

N2 - Background: Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and havemultiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing,together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to beputatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of theseenzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes.Results: We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substratesthat can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substratesof biomass materials in which the complexity of plant cell walls is partially maintained.Conclusions: We show that these substrates can be used to screen the activities of glycosyl hydrolases, lyticpolysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. Weenvisage that the assays we have developed will be used primarily for first-level screening of large numbers of putativecarbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated roboticenzyme screening systems

AB - Background: Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and havemultiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing,together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to beputatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of theseenzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes.Results: We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substratesthat can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substratesof biomass materials in which the complexity of plant cell walls is partially maintained.Conclusions: We show that these substrates can be used to screen the activities of glycosyl hydrolases, lyticpolysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. Weenvisage that the assays we have developed will be used primarily for first-level screening of large numbers of putativecarbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated roboticenzyme screening systems

U2 - 10.1186/s13068-015-0250-y

DO - 10.1186/s13068-015-0250-y

M3 - Journal article

C2 - 25969695

VL - 8

JO - Biotechnology for Biofuels

JF - Biotechnology for Biofuels

SN - 1754-6834

M1 - 70

ER -