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Author:
Date:
2014-05-05
[Abstract] Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide a detailed protocol using high performance liquid chromatography (HPLC) and ...
[摘要] 氨基酸消旋酶是在可逆反应中反转氨基酸（AA）的α-碳立体化学，在它们的L-和D-对映异构体之间相互转化氨基酸的酶。在细菌中，已知它们具有分解代谢的生理功能，但也参与许多D-AAs的合成，包括D-谷氨酸和D-丙氨酸，它们是细菌细胞壁的肽聚糖层的必需组分。因此，氨基酸消旋酶代表了杀菌化合物开发的重要目标。氨基酸消旋酶也被生物技术工业视为用于生产经济相关D-AAs的重要催化剂。在这里，我们提供使用高效液相色谱法（HPLC）和1-氟-2,4-二硝基苯基-5-L-丙氨酰胺（FDAA，也是Marfey的试剂）的新型氨基酸消旋酶的表征的详细协议。本文所述的方案被设计为获得准确的动力学参数（k cat cat，K M max值）。优化酶浓度和反应时间，以使反向反应最小化，这可在测定消旋酶反应时混淆结果。
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Author:
Date:
2013-12-05
[Abstract] Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. Previous biochemical and proteomic studies have revealed that the Gram-negative bacteria-derived OMVs are composed of various components like outer membrane proteins, lipopolysaccharides, outer membrane lipids, periplasmic proteins, DNA, and RNA. Here, in this protocol, we describe the method to isolate the OMVs from the culture supernatant of Escherichia coli (E. coli).
[摘要] 外膜囊泡（OMV）是从所有革兰氏阴性细菌和革兰氏阳性细菌产生的20-200nm的球形双层磷脂，迄今为止研究。 以前的生物化学和蛋白质组学研究已经显示，革兰氏阴性细菌衍生的OMV由各种组分组成，如外膜蛋白，脂多糖，外膜脂质，周质蛋白，DNA和RNA。 在这里，在该方案中，我们描述了从大肠杆菌的培养物上清液中分离OMV的方法（大肠杆菌）。
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