HPLC Analysis of Secreted Organic Acids
高效液相色谱法（HPLC）分析分泌有机酸
发布: 2016年04月20日第6卷第8期 DOI: 10.21769/BioProtoc.1786 浏览次数: 11380
评审: Maria SinetovaElizabeth LibbyRoman A. Sidorov
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参见作者原研究论文
The authors used this protocol in:
Sep 2015
Abstract
Under certain growth conditions some microorganisms secrete organic acids into the extracellular medium to relieve the accumulation of excess energy carriers, and/or to reduce toxic concentrations of organic acids. For example, a glycogen-deficient ∆glgC mutant of the cyanobacterium Synechococcus sp. PCC 7002 secretes pyruvate, acetate, α-ketoglutarate, α-ketoisocaproate and succinate (Davies et al., 2014; Jackson et al., 2015). Secretion of these organic acids functions as a putative energy-spilling mechanism in the absence of glycogen, the major carbon and reductant sink in this organism. Identification of secreted organic acids can facilitate the design of metabolic engineering strategies that funnel over-accumulating organic acids towards metabolic pathways that make a product of interest (such as a biofuel). Here, we describe a method for analyzing secreted organic acids in the extracellular media using high-performance liquid chromatography (HPLC). This method was developed for analysis of organic acids secreted by photosynthetic microbes (cyanobacteria and algae) into media, but could be used to analyze organic acids secreted by any microorganism cultivated in liquid medium.
Keywords: Organic acids (有机酸)
Microorganism (微生物)
HPLC (高效液相色谱法)
Secreted metabolites (分泌的代谢产物)
Metabolism (代谢)
Materials and Reagents
- 2 ml Eppendorf tubes (VWR international, catalog number: 20170-170 )
- PIPETMAN Classic P1000 pipette (Gilson Scientific Ltd., catalog number: F123602 )
- P1000 pipette tips (VWR international, catalog number: 83007-376 )
- 1 ml disposable syringe (BD, catalog number: 309659 )
- Syringe filter, 0.45 µm PTFE membrane (Pall Corporation, catalog number: PN4543 )
- Disposable filter unit (0.45 µm) (Thermo Fisher Scientific, NalgeneTM, catalog number: 166-0045 )
- Screw-thread chromatography vials (VWR International, catalog number: 66009-858 )
- Cyanobacterium Synechococcus sp. PCC 7002
- Liquid culture (cyanobacteria or other microorganism)
- Sulfuric acid (H2SO4) (Merck Millipore Corporation, catalog number: SX1244 )
- Succinate (Sigma-Aldrich, catalog number: S3674 )
- α-ketoglutarate acid (Sigma-Aldrich, catalog number: 75890 )
- Acetic acid (Sigma-Aldrich, catalog number: 338826 )
- Pyruvate (Sigma-Aldrich, catalog number: 107360 )
- α-ketoisocaproate (Sigma-Aldrich, catalog number: 68255 )
Note: It is also named “4-Methyl-2-oxovaleric acid” on Sigma-Aldrich website. - 8 mM H2SO4 (see Recipes)
- 50 mM succinate stock solution (see Recipes)
- 50 mM α-ketoglutarate stock solution (see Recipes)
- 50 mM acetic acid stock solution (see Recipes)
- 50 mM pyruvate stock solution (see Recipes)
- 50 mM α-ketoisocaproate stock solution (see Recipes)
- 10 mM organic acids standard mixture (see Recipes)
Equipment
- Microcentrifuge (Beckman Coulter, catalog number: B30147 )
- Surveyor Plus HPLC (Thermo Fisher Scientific) composed of
- Surveyor LC pump
- Surveyor Autosampler (AS)
- Surveyor Photo Diode Array (PDA) Plus detector
- Surveyor Refractive Index (RI) Plus detector
- Aminex fermentation monitoring column (150 mm by 7.8 mm), stationary phase Polystyrene-divinylbenzene sulfonic acid resin, 9 μM particle size, 8% cross linkage (Bio-Rad Laboratories, catalog number: 1250115 )
- Micro-Guard Cation H Cartridge guard column, 30 x 4.6 mm, hydrogen form, pH range 1-3, for Aminex® hydrogen-form columns (Bio-Rad Laboratories, catalog number: 1250129 )
- Surveyor LC pump
- ChromQuest™ Software Platform (Thermo Fisher Scientific, catalog number: INQSOF012 )
Software
- Chromquest software
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Davies, F. K., D’Adamo, S. and Posewitz, M. C. (2016). HPLC Analysis of Secreted Organic Acids. Bio-protocol 6(8): e1786. DOI: 10.21769/BioProtoc.1786.
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分类
生物化学 > 其它化合物 > 酸
微生物学 > 微生物生物化学 > 其它化合物
微生物学 > 微生物新陈代谢 > 其它化合物
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