| Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining
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Author:
Date:
2018-03-05
[Abstract] Ralstonia eutropha H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granules by fluorescence microscopy qualitatively. Our second approach enables the conversion of PHB to volatile hydroxycarboxylic acid methyl esters by acidic methanolysis and their quantification by gas chromatography. Through this method, it is possible to obtain an absolute quantification of PHB, e.g., per cell dry weight.
[摘要] 在生长过程中，富养罗尔斯通氏菌H16产生和动员（重新利用）细胞内聚羟基丁酸酯（PHB）颗粒。 该协议描述了细胞内尼罗红染色的PHB颗粒的可视化和通过气相色谱定量PHB。 我们的第一种方法描述了如何通过荧光显微镜定性分析PHB颗粒。 我们的第二种方法可以通过酸性甲醇分解和气相色谱定量法将PHB转化为挥发性羟基羧酸甲酯。 通过该方法，可以获得PHB的绝对定量，例如，每细胞干重。
【背景】聚羟基脂肪酸酯（PHA），尤其是聚羟基丁酸酯（PHB），是许多原核生物物种中的能量和碳储存化合物，确保细菌在压力条件下存活（Anderson和Dawes，1990;Pötter和Steinbüchel，2006; Jendrossek和Pfeiffer，2014; Bresan， >等。，2016）。这些生物聚合物的工业应用是生物降解塑料的生产（Chen，2009; Riedel等人，2015）和潜在药物成分的研究（Wu，2009; Zonari等人， ，2015; Pacheco et al。，2015; Giretova ...
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| Assays for the Detection of Rubber Oxygenase Activities
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Author:
Date:
2017-03-20
[Abstract] Microbial biodegradation of rubber relies on extracellular rubber oxygenases that catalyze the oxidative cleavage of the double bond of the polyisoprene backbone into oligo-isoprenoids. This protocol describes the determination of rubber oxygenase activities by an online measurement of molecular oxygen consumption via a non-invasive fluorescence-based assay. The produced oligo-isoprenoid cleavage products with terminal keto- and aldehyde-groups are identified qualitatively and quantitatively by HPLC. Our method allows for the characterization of homologue rubber oxygenases, and can likely be adapted to assay other oxygenases consuming dioxygen. Here we describe the determination of rubber oxygenase activities at the examples of the so far two known types of rubber oxygenases, ...
[摘要] 橡胶的微生物生物降解依赖于细胞外橡胶加氧酶，其催化聚异戊二烯骨架的双键氧化裂解成低聚异戊二烯。该方案描述了通过基于非侵入性荧光的测定在线测量分子氧消耗来测定橡胶加氧酶活性。通过HPLC定性和定量鉴定产生具有末端酮基和醛基的寡聚异戊二烯切割产物。我们的方法允许表征同系物橡胶加氧酶，并且可能适用于测定消耗双氧的其他加氧酶。在这里，我们描述了迄今为止两种已知类型的橡胶加氧酶，即橡胶加氧酶A（RoxA）和胶乳清除蛋白（Lcp）的实例的橡胶加氧酶活性的测定。
背景 ...
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| Extraction and Quantification of Sphingosine 1-Phosphate (S1P)
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Author:
Date:
2016-05-20
[Abstract] Sphingosine 1-phosphate (S1P) is a lipid metabolite and signaling molecule involved in many different physiological processes including lymphocyte circulation, T cell differentiation, antigen presentation, and maintenance of the vascular endothelial barrier. S1P is a ligand of five different G protein-coupled cell surface receptors designated S1P1-5. It has also been described as an intracellular second messenger. Quantification of S1P in biological samples is therefore an important task to decipher its signaling capabilities in vivo under physiological and pathophysiological conditions in different body fluids and organs. In this protocol, quantification of S1P is performed by liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-MS/MS).
[摘要] 鞘氨醇1-磷酸（S1P）是参与许多不同生理过程的脂质代谢物和信号分子，所述生理过程包括淋巴细胞循环，T细胞分化，抗原呈递和血管内皮屏障的维持。 S1P是称为S1P 1-5的五种不同的G蛋白偶联的细胞表面受体的配体。 它也被描述为细胞内第二信使。 因此，生物样品中S1P的定量是在不同体液和器官中的生理和病理生理条件下破译其体内信号传导能力的重要任务。 在该方案中，通过耦合到三重四极杆质谱（LC-MS/MS）的液相色谱法进行S1P的定量。
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