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HPLC Analysis of Secreted Organic Acids
高效液相色谱法(HPLC)分析分泌有机酸   

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本实验方案简略版
Applied and Environmental Microbiology
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

  1. 2 ml Eppendorf tubes (VWR international, catalog number: 20170-170 )
  2. PIPETMAN Classic P1000 pipette (Gilson Scientific Ltd., catalog number: F123602 )
  3. P1000 pipette tips (VWR international, catalog number: 83007-376 )
  4. 1 ml disposable syringe (BD, catalog number: 309659 )
  5. Syringe filter, 0.45 µm PTFE membrane (Pall Corporation, catalog number: PN4543 )
  6. Disposable filter unit (0.45 µm) (Thermo Fisher Scientific, NalgeneTM, catalog number: 166-0045 )
  7. Screw-thread chromatography vials (VWR International, catalog number: 66009-858 )
  8. Cyanobacterium Synechococcus sp. PCC 7002
  9. Liquid culture (cyanobacteria or other microorganism)
  10. Sulfuric acid (H2SO4) (Merck Millipore Corporation, catalog number: SX1244 )
  11. Succinate (Sigma-Aldrich, catalog number: S3674 )
  12. α-ketoglutarate acid (Sigma-Aldrich, catalog number: 75890 )
  13. Acetic acid (Sigma-Aldrich, catalog number: 338826 )
  14. Pyruvate (Sigma-Aldrich, catalog number: 107360 )
  15. α-ketoisocaproate (Sigma-Aldrich, catalog number: 68255 )
    Note: It is also named “4-Methyl-2-oxovaleric acid” on Sigma-Aldrich website.
  16. 8 mM H2SO4 (see Recipes)
  17. 50 mM succinate stock solution (see Recipes)
  18. 50 mM α-ketoglutarate stock solution (see Recipes)
  19. 50 mM acetic acid stock solution (see Recipes)
  20. 50 mM pyruvate stock solution (see Recipes)
  21. 50 mM α-ketoisocaproate stock solution (see Recipes)
  22. 10 mM organic acids standard mixture (see Recipes)

Equipment

  1. Microcentrifuge (Beckman Coulter, catalog number: B30147 )
  2. Surveyor Plus HPLC (Thermo Fisher Scientific) composed of
    1. Surveyor LC pump
    2. Surveyor Autosampler (AS)
    3. Surveyor Photo Diode Array (PDA) Plus detector
    4. Surveyor Refractive Index (RI) Plus detector
    5. 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 )
    6. 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 )
  3. ChromQuest™ Software Platform (Thermo Fisher Scientific, catalog number: INQSOF012 )

Software

  1. Chromquest software

Procedure

  1. Sample preparation
    1. Using a pipette, remove a 2 ml aliquot from the liquid culture, place in a 2 ml Eppendorf tube, and centrifuge at 13,000 x g for 10 min to pellet the cells.
    2. Remove and keep the supernatant; this is the extracellular medium that contains secreted organic acids. At this point the supernatant may be frozen at -20 °C for later HPLC analysis or analyzed immediately as described in the following steps.
    3. Filter 500-700 µl of the sample supernatant into chromatography vials using a 1 ml disposable syringe and a 0.45 µm-pore-size filter to remove particles that may interfere with the organic acid detection and potentially clog values and lines. Repeat individually for each organic acid standard sample that will be used to construct the standard curve. Cap the vials. These are now ready for HPLC analysis.

  2. HPLC procedure
    1. Use 8 mM H2SO4 as the mobile phase for isocratic elution.
    2. Sequentially increase the column temperature to 35 °C and then 45 °C using the Chromquest software (use a mobile phase flow rate of 0.1 ml min-1). Set the Refractive Index Detector to 50 °C.
    3. Once the column has reached 45 °C, download a method for measuring samples with the following settings. These settings are a standard protocol for the fermentation monitoring column; the only optimization is the increased run time to ensure the sample has completely eluted from the column.
      1. Surveyor LC pump: total flow, 0.5 ml/min; pressure limits, 0-1,100 psi; run time, 40 min.
      2. Surveyor PDA Plus: run time, 40 min; scans, 200-360 nm, scan rate, 1.0 Hz; bandwidth 1 nm.
      3. Surveyor AS: injection volume, 25 µl; needle height from bottom, 2.0 mm; syringe speed, 8 µl sec-1; flush speed, 70 µl sec-1; flush volume, 500 µl; wash volume, 300 µl; flush/wash source, 8 mM H2SO4 mobile phase; set tray temperature to 10 °C; enable column oven control temperature to 45 °C.
      4. Surveyor RI Plus: run time, 40 min; data rate, 10 Hz; temperature control set to 50 °C.
    4. Wait 10 min for baseline to stabilize, place vials in the autosampler tray, create a new sequence file for your run, autozero the PDA Plus and RI detectors, and start the run.

  3. Data analysis
    1. Typical chromatograms generated by the PDA Plus detector are shown in Figure 1. Similar chromatograms are generated by the RI detector. The organic acids secreted by different strains (Figure 1A) can be identified by comparing retention times to those of known standards (Figure 1B).
    2. Use the Chromquest software to integrate and quantify the area under each organic acid peak.
    3. Create a standard curve for each organic acid by plotting the peak area vs the known concentration. Example calibration curves are shown in Figure 2.
    4. Use the standard curves to calculate the concentrations of each organic acid in the supernatant samples. This will give the concentration of organic acid present in the extracellular media. Data can be normalized to cell count to determine the amount of organic acids secreted per cell.


    Figure 1. HPLC chromatograms generated by the PDA detector. A. Extracellular media from wild-type and ∆glgC cells. B. 4 mM mixture of organic acids standards. The retention times for each organic acid using our experimental procedure are as follows: α-ketoglutarate, 5.74 min; pyruvate, 6.52 min; succinate, 7.36 min; acetate, 9.51 min; α-ketoisocaproate, 10.45 min.


    Figure 2. Example calibration curves constructed by measuring the peak area of varying concentrations of organic acid standards. A. Calibration curve obtained using PDA detector. B. Calibration curve obtained using RI detector.

Notes

  1. Liquid cultures must be of sufficient cell density to produce detectable concentrations of organic acids in the extracellular media. For example, cyanobacterial cultures were inoculated at a dry weight of 0.3 g L-1, and sampled anywhere between a dry weight of 0.3 g L-1 to 2.0 g L-1 for our experiments.
  2. Both the PDA and RI detectors will detect organic acids and so chromatograms from either detector may be used for organic acid analysis; however, each detector has specific advantages and disadvantages. RI detectors are often preferred because they have universal detection capabilities and respond to the bulk property of the analyte, i.e. its refractive index. However, one limitation of an RI detector is a lack of sensitivity. UV-Vis detectors (such as PDA) are popular because they are a simple, reliable, and sensitive detector. However, PDA detectors are unable to detect most alcohols often a co-secreted metabolite.
  3. Standards may be run individually (in addition to the standard-curve mixtures) to identify the specific retention time for each organic acid.

Recipes

  1. 8 mM H2SO4 (pH 2)
    0.898 ml H2SO4 (98%)
    Add dH2O to 1,000 ml
    Filter with 0.45 µm filter unit
  2. 50 mM succinate (stock solution, store frozen)
    14.76 mg succinate
    Add dH2O to 25 ml
  3. 50 mM α-ketoglutarate (stock solution, store frozen)
    18.26 mg α-ketoglutarate
    Add dH2O to 25 ml
  4. 50 mM acetic acid (stock solution, store frozen)
    71.4 µl acetic acid
    Add dH2O to 25 ml
  5. 50 mM pyruvate (stock solution, store frozen)
    88.65 µl pyruvate (98%)
    Add dH2O to 25 ml
  6. 50 mM α-ketoisocaproate (stock solution, store frozen)
    154.19 µl α-ketoisocaproate (>98%)
    Add dH2O to 25 ml
  7. 10 mM organic acids standard mixture (store frozen)
    Mix 2 ml of each of the five 50 mM organic acid standards above to give a 10 mM solution of mixed organic acids (final volume 10 ml).
    Perform a serial dilution of this mixed organic acid solution to generate a range of concentrations appropriate for constructing a standard curve; for example, 5 mM, 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.1 mM. The range of concentrations of the standard curve should span those concentrations measured in the extracellular samples.

Acknowledgments

This protocol was adapted from the previously published study, D’Adamo et al. (2014) and it was performed by Davies et al. (2014) and Jackson et al. (2015). This work was supported by the US Air Force Office of Scientific Research (grant AFOSR 9550-14-1-0147).

References

  1. Davies, F. K., Work, V. H., Beliaev, A. S. and Posewitz, M. C. (2014). Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp. PCC 7002. Front Bioeng Biotechnol 2: 21.
  2. D'Adamo, S., Jinkerson, R. E., Boyd, E. S., Brown, S. L., Baxter, B. K., Peters, J. W. and Posewitz, M. C. (2014). Evolutionary and biotechnological implications of robust hydrogenase activity in halophilic strains of Tetraselmis. PLoS One 9(1): e85812.
  3. Jackson, S. A., Eaton-Rye, J. J., Bryant, D. A., Posewitz, M. C. and Davies, F. K. (2015). Dynamics of photosynthesis in a glycogen-deficient glgC mutant of Synechococcus sp. Strain PCC 7002. Appl Environ Microbiol 81(18): 6210-6222.

简介

在某些生长条件下,一些微生物将有机酸分泌到细胞外介质中以减轻过量能量载体的积累,和/或降低有机酸的有毒浓度。例如,蓝细菌聚集球菌属的糖原缺陷型ΔemggC 突变体。 PCC 7002分泌丙酮酸盐,乙酸盐,α-酮戊二酸盐,α-酮异己酸盐和琥珀酸盐(Davies等人,2014; Jackson等人,2015)。这些有机酸的分泌在不存在糖原的情况下作为推定的能量溢出机制起作用,主要碳和还原剂在该生物体中下沉。分泌的有机酸的鉴定可以促进代谢工程策略的设计,其将过量积聚的有机酸朝向产生感兴趣的产物(例如生物燃料)的代谢途径。在这里,我们描述了使用高效液相色谱(HPLC)分析胞外介质中分泌的有机酸的方法。该方法被开发用于分析由光合微生物(蓝细菌和藻类)分泌到培养基中的有机酸,但是可以用于分析在液体培养基中培养的任何微生物分泌的有机酸。

关键字:有机酸, 微生物, 高效液相色谱法, 分泌的代谢产物, 代谢

材料和试剂

  1. 2ml Eppendorf管(VWR international,目录号:20170-170)
  2. PIPETMAN Classic P1000移液管(Gilson Scientific Ltd.,目录号:F123602)
  3. P1000移液枪吸头(VWR国际,目录号:83007-376)
  4. 1ml一次性注射器(BD,目录号:309659)
  5. 注射器过滤器,0.45μmPTFE膜(Pall Corporation,目录号:PN4543)
  6. 一次性过滤器单元(0.45μm)(Thermo Fisher Scientific,Nalgene TM ,目录号:166-0045)
  7. 螺纹色谱小瓶(VWR International,目录号:66009-858)
  8. 蓝藻属(Cyanobacterium)聚球藻属PCC 7002
  9. 液体培养(蓝细菌或其他微生物)
  10. 硫酸(H 2 SO 4)(Merck Millipore Corporation,目录号:SX1244)
  11. 琥珀酸酯(Sigma-Aldrich,目录号:S3674)
  12. α-酮戊二酸(Sigma-Aldrich,目录号:75890)
  13. 乙酸(Sigma-Aldrich,目录号:338826)
  14. 丙酮酸(Sigma-Aldrich,目录号:107360)
  15. α-酮异己酸盐(Sigma-Aldrich,目录号:68255) 注意:在Sigma-Aldrich网站上也称为"4-甲基-2-氧代戊酸"。
  16. 8 mM H 2 SO 4子(参见配方)
  17. 50 mM琥珀酸盐储备溶液(见配方)
  18. 50 mMα-酮戊二酸储备液(见配方)
  19. 50 mM乙酸储备溶液(见配方)
  20. 50 mM丙酮酸储备溶液(见配方)
  21. 50 mMα-酮异己酸盐储备液(见配方)
  22. 10 mM有机酸标准混合物(见配方)

设备

  1. 微量离心机(Beckman Coulter,目录号:B30147)
  2. Surveyor Plus HPLC(Thermo Fisher Scientific)由
    组成
    1. 测量仪液位泵
    2. 测量员自动进样器(AS)
    3. 测量器光电二极管阵列(PDA)Plus检测器
    4. 测量仪折射率(RI)Plus检测器
    5. Aminex发酵监测柱(150mm×7.8mm),静止 ?相聚苯乙烯 - 二乙烯基苯磺酸树脂,9μM颗粒 大小,8%交联(Bio-Rad Laboratories,目录号:1250115)
    6. 微保护阳离子H柱保护柱,30×4.6mm,氢气 形式,pH范围1-3,对于Aminex型氢型柱(Bio-Rad 实验室,目录号:1250129)
  3. ChromQuest?软件平台(Thermo Fisher Scientific,目录号:INQSOF012)

软件

  1. Chromquest软件

程序

  1. 样品准备
    1. 使用移液管,从液体培养基中取出2ml等分试样,放置 ?在2ml Eppendorf管中,并以13,000×g离心10分钟 沉淀细胞
    2. 取出并保留上清液,这是 包含分泌的有机酸的胞外培养基。在此刻 ?可将上清液在-20℃冷冻用于以后的HPLC分析或 立即分析,如以下步骤所述。
    3. 过滤 将500-700μl样品上清液加入到色谱小瓶中 ?ml一次性注射器和0.45μm孔径过滤器以除去 可能干扰有机酸检测的颗粒 潜在堵塞值和线。对每个有机物重复单独 酸标准样品,用于构建标准曲线。 盖住小瓶。现在可以进行HPLC分析了。

  2. HPLC操作
    1. 使用8mM H 2 SO 4作为用于等度洗脱的流动相。
    2. 依次将柱温升至35℃,然后升至45℃ 使用Chromquest软件(使用0.1ml的流动相流速 min -1 )。将折射率检测器设置为50°C。
    3. 一旦 柱已达到45°C下载一种测量样品的方法 以下设置。这些设置是一个标准协议 发酵监测柱,唯一的优化就是增加 运行时间以确保样品已完全从柱中洗脱。
      1. Surveyor LC泵:总流量,0.5 ml/min;压力极限,0-1100 psi;运行时间,40分钟。
      2. Surveyor PDA Plus:运行时间,40分钟;扫描,200-360nm,扫描速率,1.0Hz;带宽1 nm。
      3. 测量员AS:注射体积,25μl;针头高度从底部,2.0 ?mm;注射器速度,8μlsec -1 -1 ;冲洗速度,70μlsec -1 -1 ;冲洗量, 500μl;洗涤体积,300μl;冲洗/洗涤源,8mM H 2 SO 4 4流动相; ?将托盘温度设置为10°C;启用柱温箱控制温度 至45℃
      4. 测量员RI Plus:运行时间,40分钟;数据速率,10Hz;温度控制设置为50°C。
    4. 等待10分钟使基线稳定,将小瓶放入 自动进样器托盘,为您的运行创建新的序列文件,自动调零 PDA Plus和RI检测器,并启动运行。

  3. 数据分析
    1. 由PDA Plus检测器产生的典型色谱图如图所示 ?图1. RI检测器产生类似的色谱图。的 由不同菌株分泌的有机酸(图1A)可以 通过将保留时间与已知标准的保留时间进行比较来鉴 (图1B)。
    2. 使用Chromquest软件对每个有机酸峰下的面积进行积分和量化
    3. 通过绘制峰值为每个有机酸创建一个标准曲线 面积对已知浓度。示例校准曲线如图所示 ?图2.
    4. 使用标准曲线计算 上清液样品中每种有机酸的浓度。这个 将得到存在于细胞外的有机酸的浓度 ?媒体。数据可以归一化为细胞计数以确定量 每个细胞分泌的有机酸。


    图1. PDA检测器产生的HPLC色谱图。 A.来自野生型和ΔggC/C细胞的细胞外培养基。 B. 4mM有机酸标准品混合物。使用我们的实验程序,每种有机酸的保留时间如下:α-酮戊二酸盐,5.74分钟;丙酮酸盐,6.52分钟;琥珀酸酯,7.36分钟;乙酸乙酯,9.51分钟; α-酮异己酸盐,10.45分钟

    图2.通过测量不同浓度的有机酸标准品的峰面积构建的示例校准曲线。 A.使用PDA检测器获得的校准曲线。 B.使用RI检测器获得的校准曲线

笔记

  1. 液体培养物必须具有足够的细胞密度以在胞外培养基中产生可检测浓度的有机酸。例如,以0.3g L -1的干重接种蓝细菌培养物,并且在干重0.3g L -1至2.0g L -1之间取样, sup> -1 。
  2. PDA和RI检测器都将检测有机酸,因此来自任一检测器的色谱图可用于有机酸分析;然而,每个检测器具有特定的优点和缺点。 RI检测器通常是优选的,因为它们具有通用的检测能力并响应于分析物的整体性质,即其折射率。然而,RI检测器的一个限制是灵敏度的缺乏。 UV-Vis检测器(例如PDA)是受欢迎的,因为它们是简单,可靠和灵敏的检测器。然而,PDA检测器不能检测大多数醇,通常是共同分泌的代谢物
  3. 标准品可以单独运行(除了标准曲线混合物),以确定每种有机酸的比保留时间

食谱

  1. 8mM H 2 SO 4(pH 2) 0.898ml H 2 SO 4(98%)
    将dH <2> O添加至1,000 ml
    用0.45μm过滤器单元过滤
  2. 50 mM琥珀酸盐(储备溶液,冷冻保存)
    14.76mg琥珀酸盐
    将dH <2> O加到25ml
  3. 50 mMα-酮戊二酸(储备液,冷冻保存)
    18.26mgα-酮戊二酸盐 将dH <2> O加到25ml
  4. 50 mM乙酸(储备液,冷冻保存)
    71.4μl乙酸
    将dH <2> O加到25ml
  5. 50 mM丙酮酸(储备液,冷冻保存)
    88.65μl丙酮酸(98%) 将dH <2> O加到25ml
  6. 50mMα-酮异己酸盐(储备溶液,冷冻保存)
    154.19μlα-酮异己酸盐(> 98%)
    将dH <2> O加到25ml
  7. 10mM有机酸标准混合物(冷冻保存)
    将上述5种50mM有机酸标准品各2ml混合,得到10mM混合有机酸溶液(最终体积10ml)。
    对该混合有机酸溶液进行连续稀释以产生适于构建标准曲线的一系列浓度;例如5mM,2mM,1mM,0.5mM,0.25mM,0.1mM。标准曲线的浓度范围应该跨越在细胞外样品中测量的浓度

致谢

该协议改编自先前发表的研究(D'Adamo等人)(2014),并且由Davies等人(2014)和Jackson等人进行。 et al。(2015)。这项工作是由美国空军科学研究办公室(授予AFOSR 9550-14-1-0147)支持。

参考文献

  1. Davies,F. K.,Work,V. H.,Beliaev,A.S。和Posewitz,M.C。(2014)。 在野生型中工程苎烯和bisabolene产生和聚球藻属的糖原 - em> sp。 PCC 7002. Front Bioeng Biotechnol 2:21.
  2. D'Adamo,S.,Jinkerson,R.E.,Boyd,E.S.,Brown,S.L.,Baxter,B.K.,Peters,J.W.and Posewitz,M.C。(2014)。 稳定的氢化酶活性在嗜盐菌属Tetraselmis中的进化和生物技术影响。 PLoS One 9(1):e85812。
  3. Jackson,S.A.,Eaton-Rye,J.J.,Bryant,D.A.,Posewitz,M.C.and Davies,F.K.(2015)。 聚球藻属的糖原 - 基因glgC突变体中的光合作用动力学。 Strain PCC 7002. Appl Appl Environ Microbiol 81(18):6210-6222。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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