High-Throughput In Vitro Screening for Inhibitors of Cereal α-Glucosidase

Research output: Chapter in Book/Report/Conference proceedingBook chapter – Annual report year: 2018Researchpeer-review

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The hydrolysis of starch is a key step in plant germination, which also has relevance in the malting and brewing processes for beer and spirit production. Gaps in knowledge about this metabolic process exist that cannot easily be addressed using traditional genetic techniques, due to functional redundancy in many of the enzyme activities required for alpha-glucan metabolism in cereal crop species. Chemical inhibitors provide opportunities to probe the role of carbohydrate-active enzymes and the phenotypes associated with inhibition of specific enzymes. Iminosugars are the largest group of carbohydrate-active enzyme inhibitors and represent an underused resource for the dissection of plant carbohydrate metabolism. Herein we report a method for carrying out a reverse chemical genetic screen on α-glucosidase, the enzyme that catalyzes the final step in starch degradation during plant germination, namely the hydrolysis of maltose to release glucose. This chapter outlines the use of a high-throughput screen of small molecules for inhibition of α-glucosidase using a colorimetric assay which involves the substrate p-nitrophenyl α-D-glucopyranoside. Identified inhibitors can be further utilized in phenotypic screens to probe the roles played by amylolytic enzymes. Furthermore this 96-well plate-based method can be adapted to assay exo-glycosidase activities involved in other aspects of carbohydrate metabolism.
Original languageEnglish
Title of host publicationPlant Chemical Genomics - Methods and Protocols
EditorsFriedrich Fauser, Martin Jonikas
PublisherSpringer
Publication date2018
Pages101-115
Chapter9
ISBN (Print)978-1-4939-7873-1
ISBN (Electronic)978-1-4939-7874-8
DOIs
Publication statusPublished - 2018
SeriesMethods in Molecular Biology
Volume1795
ISSN1064-3745
CitationsWeb of Science® Times Cited: No match on DOI

    Research areas

  • Barley, Carbohydrate-active enzyme, Cereal, Chemical screen, Deoxynojirimycin, Enzyme inhibition, Germination, Iminosugar, Starch, α-Glucosidase

ID: 149069650