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Rugen, Michael D., Rejzek, Martin, Naested, Henrik, Svensson, Birte and Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275
(2018)
High-throughput in vitro screening for inhibitors of cereal α-glucosidase.
In:
Methods in Molecular Biology.
Methods in Molecular Biology
.
Humana Press Inc, pp. 101-115.
Abstract
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.
| Item Type: | Book Section |
|---|---|
| Additional Information: | Funding Information: The authors would like to thank Alison M Smith and Vasilios Andriotis for helpful discussions and advice. This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC, UK) Institute Strategic Programme Grant (MET) [BB/J004561/1] to the John Innes Centre, a BBSRC-Crop Improvement Research Club (CIRC) grant BB/I017291/1 to A.M.S. and R.A.F, and BBSRC PhD studentship BB/ J500069/1 to M.D.R. Publisher Copyright: © 2018, Springer Science+Business Media, LLC, part of Springer Nature. |
| Uncontrolled Keywords: | barley,carbohydrate-active enzyme,cereal,chemical screen,deoxynojirimycin,enzyme inhibition,germination,iminosugar,starch,α-glucosidase,molecular biology,genetics ,/dk/atira/pure/subjectarea/asjc/1300/1312 |
| Faculty \ School: | Faculty of Science > School of Chemistry, Pharmacy and Pharmacology |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 04 Sep 2024 08:30 |
| Last Modified: | 24 Sep 2024 08:28 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96496 |
| DOI: | 10.1007/978-1-4939-7874-8_9 |
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