High-Throughput In Vitro Screening for Inhibitors of Cereal a-Glucosidase.
High-Throughput In Vitro Screening for Inhibitors of Cereal a-Glucosidase.
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谷物α-葡萄糖苷酶抑制剂的高通量体外筛选。
DOI：
10.1007/978-1-4939-7874-8_9
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Rugen MD
中科院分区：
文献类型：
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作者：
Rugen MD
淀粉的水解是植物发芽的关键步骤，其在啤酒和烈酒生产的麦芽和酿造过程中也具有相关性。由于谷类作物中α-葡聚糖代谢所需的许多酶活性存在功能冗余，因此，对这一代谢过程的认识存在空白，无法通过传统遗传技术轻松解决。化学抑制剂提供了探索碳水化合物活性酶的作用和与特定酶的抑制相关的表型的机会。亚氨基糖是碳水化合物活性酶抑制剂中最大的一类，是研究植物碳水化合物代谢的一种未充分利用的资源。本文报道了一种对α-葡萄糖苷酶进行反向化学遗传筛选的方法，α-葡萄糖苷酶催化植物萌发过程中淀粉降解的最后一步，即水解麦芽糖释放葡萄糖。本章概述了采用比色法（包括底物对硝基苯基α-d-吡喃葡萄糖苷）高通量筛选抑制α-葡萄糖苷酶的小分子。鉴定的抑制剂可进一步用于表型筛选以探测淀粉分解酶所起的作用。此外，这种基于96孔板的方法可以适用于测定参与碳水化合物代谢的其他方面的外切糖苷酶活性。
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.