Colorimetric assays for the detection of C4- and C2-epimerization of NDP-glucose to NDP-galactose and NDP-mannose

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry Pub Date : 2025-07-18 DOI:10.1016/j.ab.2025.115947

Ulrike Vogel, Renger Dijkstra, Koen Beerens, Tom Desmet

引用次数: 0

Abstract

CDP-tyvelose 2-epimerase-like enzymes and UDP-glucose 4-epimerases belong to the Nucleotide Sugar active Short-Chain Dehydrogenase/Reductase (NS-SDR) superfamily and convert NDP-Glc to NDP-Man and NDP-Gal, respectively. The product detection is well established using HPLC-based methods, capillary electrophoresis and thin layer chromatography for both epimerization reactions. However, the lack of a reliable colorimetric assays and poor substrate availability slow down the screening process of these enzymes, for example during enzyme engineering projects. After testing 16 phenolic compounds with different aldohexoses in concentrated sulfuric acid, thymol was found suitable for the detection of (NDP-) mannose and 1-naphthol for the detection of (NDP-) galactose in the presence of (NDP-) glucose. As proof-of-concept, the optimized assay was used for the biochemical characterization of the CDP-tyvelose 2-epimerase from Ardenticatenia bacterium.

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检测ndp -葡萄糖向ndp -半乳糖和ndp -甘露糖的C4-和c2 -外映体化的比色法

CDP-tyvelose 2- epimase样酶和UDP-glucose 4- epimase属于核苷酸糖活性短链脱氢酶/还原酶（NS-SDR）超家族，分别将NDP-Glc转化为NDP-Man和NDP-Gal。采用基于高效液相色谱的方法，毛细管电泳和薄层色谱对这两种外映反应进行了很好的检测。然而，缺乏可靠的比色测定和较差的底物可用性减慢了这些酶的筛选过程，例如在酶工程项目中。在浓硫酸中检测了16种不同醛己糖的酚类化合物，发现百里香酚适用于（NDP-）甘露糖的检测，1-萘酚适用于（NDP-）葡萄糖存在下（NDP-）半乳糖的检测。作为概念验证，优化后的检测方法被用于Ardenticatenia细菌的CDP-tyvelose 2- epimase的生化表征。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Analytical biochemistry 生物-分析化学

CiteScore

5.70

自引率

0.00%

发文量

283

审稿时长

44 days

期刊介绍： The journal''s title Analytical Biochemistry: Methods in the Biological Sciences declares its broad scope: methods for the basic biological sciences that include biochemistry, molecular genetics, cell biology, proteomics, immunology, bioinformatics and wherever the frontiers of research take the field. The emphasis is on methods from the strictly analytical to the more preparative that would include novel approaches to protein purification as well as improvements in cell and organ culture. The actual techniques are equally inclusive ranging from aptamers to zymology. The journal has been particularly active in: -Analytical techniques for biological molecules- Aptamer selection and utilization- Biosensors- Chromatography- Cloning, sequencing and mutagenesis- Electrochemical methods- Electrophoresis- Enzyme characterization methods- Immunological approaches- Mass spectrometry of proteins and nucleic acids- Metabolomics- Nano level techniques- Optical spectroscopy in all its forms. The journal is reluctant to include most drug and strictly clinical studies as there are more suitable publication platforms for these types of papers.