欧洲山芥皂苷配基的提取和葡糖基化修饰

Jörg M. Augustin; Carl Erik Olsen; Søren Bak

doi:10.21769/BioProtoc.826

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Extraction and Reglucosylation of Barbarea vulgaris Sapogenins

欧洲山芥皂苷配基的提取和葡糖基化修饰

Jörg M. Augustin email

CO Carl Erik Olsen

SB Søren Bak

发布: 2013年07月20日第3卷第14期 DOI: 10.21769/BioProtoc.826 浏览次数: 10059

评审: Tie Liu

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Dec 2012

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

一种快速定量拟南芥幼苗中铁的方法

Chandan Kumar Gautam [...] Wolfgang Schmidt

2022年03月05日 3025 阅读

拟南芥根原生质体完整液泡的分离及元素分析

Chuanfeng Ju [...] Zhenqian Zhang

2023年03月05日 1223 阅读

高效液相色谱法检测培养基及微藻细胞中草甘膦、氨基甲基膦酸和抗坏血酸的含量

Juan Manuel Ostera [...] Gabriela Malanga

2025年04月05日 495 阅读

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 Barbarea vulgaris (Augustin et al., 2012). All four enzymes utilize uridine diphosphate glucose (UDP-glc) as glucosyl-moiety donor and different sapogenins such as the oleanane sapogenins oleanolic acid and hederagenin as glucosyl-moiety acceptor. Oleanolic acid and hederagenin both naturally occur in G-type B. vulgaris, where they are predominantly found in their 3-O-cellobiosylated form. Additional saponins from G-type B. vulgaris have been identified by Nielsen et al., 2010. However, the majority of saponins and sapogenins that occur in B. vulgaris remain unidentified.

Materials and Reagents

Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
Polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, catalog number: 77627 )
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
Tris(hydroxymethyl)aminomethane (Tris base) (Sigma-Aldrich, catalog number: T1503 )
Ethyl acetate (Sigmal-Aldrich, catalog number: 34972 )
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) (Sigma-Aldrich, catalog number: T5130 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
Uridine-5’-diphosphoglucose (UDP-Glc) (Sigma-Aldrich, catalog number: S451649 )
Silica gel 60 F₂₅₄ TLC plates (EMD Millipore, catalog number: 1055540001 )
Polyvinylidene difluoride (PVDF) filter plate (0.45 μm pore diameter) (EMD Millipore, catalog number: MAHVN4510 )
FRETWorks S-tag assay kit (EMD Millipore, catalog number: 70724 )

Equipment

Water bath
Centrifuge for 50 ml and 15 ml conical centrifugation tubes (VWR international, catalog number: 89004-368 )
Thermomixer (VWR international, catalog number: 21516-168 )
pH indicator paper (Whatman, catalog number: 2600-100A )
Vacuum centrifuge (Labogene, catalog number: 7.008.100.777 )
Thin layer chromatography (TLC) developing chamber (VWR international, catalog number: 21432-739 )
Aldrich flask-type sprayer (Sigma-Aldrich, catalog number: Z190373 )
Heat block (VWR international, catalog number: 12621-120 )
LC-MS analysis was carried out on an Agilent 1100 Series LC (Agilent Technologies), equipped with a Gemini NX column (Phenomenex), and coupled to a Bruker HCT-Ultra ion trap mass spectrometer (Bruker Daltonics)

Software

DataAnalysis 4.0 (Bruker Daltonics)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Augustin, J. M., Olsen, C. E. and Bak, S. (2013). Extraction and Reglucosylation of Barbarea vulgaris Sapogenins. Bio-protocol 3(14): e826. DOI: 10.21769/BioProtoc.826.
Augustin, J. M., Drok, S., Shinoda, T., Sanmiya, K., Nielsen, J. K., Khakimov, B., Olsen, C. E., Hansen, E. H., Kuzina, V., Ekstrom, C. T., Hauser, T. and Bak, S. (2012). UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance . Plant Physiol 160(4): 1881-1895.