Microscale quantitative analysis of polyhydroxybutyrate in prokaryotes using IDMS
| Autor: | Mark C.M. van Loosdrecht, S. Aljoscha Wahl, Reza M. Seifar, Mariana Itzel Velasco Alvarez, Patricia T. N. van Dam, Angela ten Pierick |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
polyhydroxybutyrate isotope dilution Serial dilution Endocrinology Diabetes and Metabolism Analytical chemistry lcsh:QR1-502 Isotope dilution Biochemistry Article lcsh:Microbiology Metabolic engineering Polyhydroxybutyrate 03 medical and health sciences Escherichia coli Sample preparation 3-hydroxy butyrate GC-MS 13C-labeled internal standard Molecular Biology Reproducibility Chromatography Chemistry 030104 developmental biology OA-Fund TU Delft Gas chromatography–mass spectrometry Quantitative analysis (chemistry) |
| Zdroj: | Metabolites, 7(2) Metabolites, Vol 7, Iss 2, p 19 (2017) Metabolites Metabolites; Volume 7; Issue 2; Pages: 19 |
| ISSN: | 2218-1989 |
| Popis: | Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled 13C-cell suspension, of an E. coli PHB producing strain, grown on U-13C-glucose as C-source. This internal 13C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/gCDW) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps. |
| Databáze: | OpenAIRE |
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