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High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

Lookup NU author(s): Professor William Willats

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


Abstract

© 2016 Journal of Visualized Experiments. Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.


Publication metadata

Author(s): Schuckel J, Kracun SK, Willats WGT

Publication type: Article

Publication status: Published

Journal: Journal of Visualized Experiments

Year: 2016

Volume: 2016

Issue: 115

Online publication date: 20/09/2016

Acceptance date: 02/04/2016

Date deposited: 11/07/2017

ISSN (electronic): 1940-087X

Publisher: Journal of Visualized Experiments

URL: http://doi.org/10.3791/54286

DOI: 10.3791/54286


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