| Product Analysis of Starch Active Enzymes by TLC
|
|
Author:
Date:
2015-10-20
[Abstract] Thin layer chromatography (TLC) is a useful technique for detecting the presence of monosaccharides through to oligosaccharides, though it needs to be optimized for the specific sugars that are analyzed. Here we present a method for visualizing the reaction product(s) of starch active enzymes, which can contain α-1, 4 linked and α-1, 6 linked glucose. This was first published in Molecular Microbiology (Cockburn et al., 2015). The TLC protocol is an adapted version of that published by Robyt and Mukerjea (Robyt and Mukerjea, 1994). For a summary of the products generated by starch active enzymes see the review by Hii et al. (2012).
[摘要] 薄层色谱（TLC）是用于检测单糖通过寡糖的存在的有用技术，尽管它需要针对所分析的特定糖进行优化。 在这里我们提出一种可视化的淀粉活性酶，可以包含α-1,4连接和α-1,6连接葡萄糖的反应产物的方法。 这首次发表于Molecular Microbiology（Cockburn等人，2015年）。 TLC方案是由Robyt和Mukerjea（Robyt和Mukerjea，1994）发表的适应版本。 对于由淀粉活性酶产生的产物的概述，参见Hii等人（2012）的综述。
|
|
|
| Determination of Enzyme Kinetic Parameters of UDP-glycosyltransferases
|
|
Author:
Date:
2013-07-20
[Abstract] The determination of enzyme kinetic parameters, such as the Km and kcat values, is an essential part of the characterization of newly discovered enzymes. This protocol describes the determination of enzyme kinetic parameters of the Barbarea vulgaris UDP-glycosyltransferases (UGTs) UGT73C11 and UGT73C13 toward the sapogenins oleanolic acid and hederagenin as sugar acceptor substrates. UGTs catalyze the transfer of glycosyl residues. They generally use uridine sugar nucleotides as their sugar donor substrates, whereas sugar acceptor substrates arise from structurally diverse sets of metabolite classes. This protocol is based on the quantification of 14C-labeled glycosides following thin layer chromatography (TLC)-based separation. The dependence of ...
[摘要] 测定酶动力学参数，如Km和kcat值，是新发现的酶的表征的重要组成部分。该方案描述了通过将Barbarea vulgaris UDP-糖基转移酶（UGTs）UGT73C11和UGT73C13的酶动力学参数作为糖受体底物朝向皂甙元酸齐墩果酸和雄蕊草素的测定。 UGTs催化糖基残基的转移。他们通常使用尿苷糖核苷酸作为其供体底物，而糖受体底物来自结构不同的代谢物类别。该方案基于以薄层色谱（TLC）为基础的分离后14C标记的糖苷的定量。测量信号对通用放射性标记的糖供体底物的依赖性允许将该方案与广泛范围的不同糖受体底物结合使用。然而，由于这里描述的TLC分离程序已被优化用于分离皂角苷及其糖苷，所以在研究其它化合物类时可能需要进行一些修饰。
|
|
|
| Extraction and Reglucosylation of Barbarea vulgaris Sapogenins
|
|
Author:
Date:
2013-07-20
[Abstract] Plants produce a vast array of natural compounds. Many of them are not commercially available, and are thus lacking to be tested as substrates for enzymes. This protocol describes the extraction and acidic hydrolysis of metabolites from Barbarea vulgaris with special focus on saponins and their agylcones (sapogenins). It was developed to determine if some B. vulgaris UDP-glucosyltransferases (UGTs) that were shown to glucosylate commercially available sapogenins, would also accept additional sapogenins from this plant as substrate, which are yet chemically uncharacterized and/or commercially unavailable (Figure 1).
Figure 1. Glucosylation reaction catalyzed by UGT73C10-UGT73C13 from ...
[摘要] 植物产生大量的天然化合物。 其中许多不是可商购的，因此缺乏作为酶的底物的测试。 该协议描述了从寻常型巴布巴属植物中提取和酸性水解代谢物，特别关注皂苷及其苷元（皂苷元）。 它被开发以确定是否有一些。 显示用于使市售皂苷元葡萄糖基化的普通UDP葡萄糖基转移酶（UGT）也将接受来自该植物的另外的皂苷元作为底物，其仍然是化学上未表征的和/或商业上不可获得的（图1）。
|
|
|