Assays for Determination of Acetylesterase Activity and Specificity Using pNP-acetyl and Acetylated Polysaccharides as Substrates

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Plant Physiology
May 2013



The acetylesterases are hydrolytic enzymes which in plants cleave acetyl groups from acetylated cell wall components, primarily polysaccharides. To estimate acetylesterase activity in plant apoplast, two assays can be used. First assay is a direct measurement of the acetylesterase activity in protein extract using synthetic substrate, pNP-acetyl. In this assay, amount of pNP released after hydrolysis of pNP-acetyl is determined by measuring the intensity of developed yellow color using spectrophotometer. The absorbance of reaction mixture is directly proportional to the activity of acetylesterases in the reaction mixture. Second assay is a determination of acetylesterase activity and its specificity towards natural polysaccharides and based on interaction between ferric perchlorate and acetyl residues resulting in ferric acetohydroxamic complex that can be quantified using spectrophotometer. In this assay, commercially available acetylated polysaccharides (xylan from Birchwood for acetylxylan esterase; pectin from citrus fruit for rhamnogalacturonan acetylesterase; or any other available polysaccharide of interest) incubated with apoplastic extract and amount of acetyl residues released from this polysaccharide is estimated using ferric perchlorate reagent (protocol was modified from McComb and McCready, 1957). The absorbance of produced colored complex is directly proportional to the amount of acetyls released from acetylated polysaccharide.

Keywords: Cell wall (细胞壁), Acetyl esterase activity (乙酰酯酶活性), Polysaccharide acetylation (多糖的乙酰化), Apoplast (质外体)

Materials and Reagents

  1. Plant material
  2. 4-Nitrophenyl acetate (Sigma-Aldrich, catalog number: N8130 )
  3. 4-Nitrophenyl (Sigma-Aldrich, catalog number: N1048 )
  4. Tris-HCl
  5. EDTA
  6. MgCl2
  7. Xylan from Birchwood (Sigma-Aldrich, catalog number: X4252 )
  8. Pectin from citrus fruit (Sigma-Aldrich, catalog number: P9436 )
  9. Sodium phosphate
  10. Sodium hydroxide
  11. Hydroxylamine hydrochloride (Acros Organics, catalog number: 5470-11-1 )
  12. Perchloric acid (Sigma-Aldrich, catalog number: 311421-50ML )
  13. Absolute methanol
  14. Glucose-penta-acetate (Sigma-Aldrich, catalog number: G2354-25G )
  15. Deionized water
  16. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 ) (optional)
  17. Extraction/reaction buffer (see Recipes)
  18. Re-suspension buffer (see Recipes)
  19. Acid-alcohol solution (see Recipes)
  20. Ferric Perchlorate reagent (MP Biomedicals, catalog number: 215875 ) (see Recipes)


  1. Sharp razor blade
  2. pH meter
  3. Microtiter plate reader
  4. Microcentrifuge
  5. Centrifuge compatible with 15 ml vials
  6. 96-well microtiter plate
  7. Parafilm
  8. Sharp razor blade
  9. 10 ml syringe
  10. Light water flow laboratory vacuum
  11. SpeedVac dryer
  12. Vortex
  13. NanoDrop spectrophotometer (optional)
  14. Shaker for microtubes


  1. Apoplast protein extraction from plant material
    Note: Acetylesterase activity assay can be applied not only for apoplast proteins but also for total proteins extracts and purified enzymes.
    1. Harvest 2-5 g of aerial parts (stems, leaves, siliques, flowers, and buds) of plants and immediately cut into 5 mm segments using a sharp razor blade.
    2. Place plant material into a 10 ml syringe which had its tip sealed with Parafilm (Figure 1A-B).
    3. Add 5 ml of pre-cooled extraction/reaction buffer. 
    4. Place the syringe into a centrifuge tube (tip is showing downwards, Figure 1C) and treat under vacuum in exicator (Figure 1D-E) twice for 15 min with a 5 min break in a cold room at 10 °C.
    5. Open the tip of syringe, carefully drain the buffer by gravity flow and discard it.
    6. Centrifuged at 1,000 x g for 10 min at 10 °C.
    7. Place apoplast extracts accumulates at the bottom of the tube on ice.
    8. Estimate protein concentration by NanoDrop or by Bradford assay described at Bradford Protein Assay (He, 2011).

      Figure 1. Apoplast extraction tools. A. Initial set: 5 ml syringe, 15 ml centrifuge tube, parafilm. B. Syringe tip sealed with parafilm. C. Syringe with sealed tip placed into centrifuge tube. D. Syringe with sealed tip in centrifuge tube placed into vacuum exicator. E. Vacuum exicator connected to the vacuum line.

  2. Total activity of acetylesterases
    1. Dilute apoplast extract with extraction/reaction buffer to obtain 0.128 mg in 100 µl.
      Note: Amount of protein for assay will vary depending on the enzymatic activity and specificity of acetylesterases present in extract. Here we perform example made on Arabidopsis extracts.
    2. Prepare blank negative control by adding of 1 μl of 200 mM pNP-acetyl to the 100 μl of boiled for 10 min apoplast extract containing 0.128 mg of protein.
    3. Add 1 μl of 200 mM pNP-acetyl to 100 µl of diluted apoplast extract.
    4. Incubate at room temperature for 0, 5, 15, 30, 60, 120, 180 min reading blank and samples absorbance at 460 nm in microplate reader at each time point.
    5. Prepare standard curve by measuring absorbance at 460 nm of 100 μl pNP solution diluted in series of concentrations ranging from 0.1 mM to 2 mM. All dilutions prepare in 3 replicates. Calculate the slope of the line which will give STD1 value equal to absorbance of 0.1 μmoles of pNP.
    6. Measure OD460 sample-OD460 blank for each time point and calculate the slope.
    7. Calculate the amount of released pNP using the following formula:
      Slope (from step B6)
      0.1 and STD1 (from step B5)
      0.128 (from step B2)
      Obtaining valueу will indicate amount of released pNP µmoles per 1 min per 1 mg of apoplast proteins

  3. Assay for acetylesterase activity using  natural substrates (cell wall derived polysaccharides)
    1. Apply 0.12 mg of apopalst protein in extraction/reaction buffer to 20 mg of natural substrate in total volume 0.6 ml.
    2. Prepare blank negative control by adding 0.12 mg of boiled apoplast protein in extraction/reaction obuffer to 20 mg of natural substrate.
    3. Incubate at room temperature for 0, 30 min, 1 h, 3 h, 6 h shaking at 130 rpm.
    4. At each time point spin down polysaccharide material (1 min at 17,000 x g) and take an aliquot 100 μl of supernatant.
      Note: After taking the aliquot vortex the pellet and continue incubation.
    5. Dry the aliquot using SpeedVac.
    6. Re-suspend dry material in 40 μl of Re-suspension buffer (use vortex).
    7. Add 100 μl of deionized water and mix carefully.
    8. Add 100 μl of acid-alcohol solution and mix carefully.
    9. Add 260 μl of Ferric Perchlorate reagent and mix carefully.
    10. Incubate at room temperature 15-30 min.
    11. Measure the absorbance at 510 nm.
    12. Measure OD510 sample-OD510 blank for each time point and calculate the slope.
    13. Prepare standard curve using glucose-penta-acetate with a series of dilutions from 0.1 mM to 2 mM in 40 μl solution following by steps C7-12 paragraphs from above. Calculate slope which will give STD2 value equal to absorbance of 0.2 nmoles of acetyl residues.
    14. Calculate the amount of released acetyls from natural substrates by following formula:
      Slope (from step C12)
      0.2 and STD2 (from step C8)
      0.02 (from a. 0.18 mg of apoplast protein was used in total 6 measurements)
      Determined value will indicate amount of released acetyls (nmoles) per 1 min per 1 mg of apoplast protein


  1. Extraction buffer
    25 mM Tris-HCl
    50 mM EDTA
    150 mM MgCl2 (pH 7.4)
  2. Re-suspension buffer
    0.25 M hydroxylamine hydrochloride
    1 M sodium hydroxide solution
  3. Acid-alcohol solution
    5% perchloric acid in absolute methanol
  4. Ferric Perchlorate reagent
    0.3% ferric perchlorate (non-yellow)
    0.5% perchloric acid dissolved in 88% methanol


The authors acknowledge Carver Funding for the support of this work.


  1. He, F. (2011). Bradford protein assay. Bio-protocol 1(6): e45.
  2. McComb, E. and McCready, R. (1957). Determination of acetyl in pectin and in acetylated carbohydrate polymers. Anal Chem 29(5): 819-821.
  3. Pogorelko, G., Lionetti, V., Fursova, O., Sundaram, R. M., Qi, M., Whitham, S. A., Bogdanove, A. J., Bellincampi, D. and Zabotina, O. A. (2013). Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens. Plant Physiol 162(1): 9-23.
  4. Pogorelko, G., Fursova, O., Lin, M., Pyle, E., Jass, J. and Zabotina, O. A. (2011). Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast. Plant Mol Biol 77(4-5): 433-445. 



关键字:细胞壁, 乙酰酯酶活性, 多糖的乙酰化, 质外体


  1. 植物材料
  2. 4-硝基苯基乙酸酯(Sigma-Aldrich,目录号:N8130)
  3. 4-硝基苯(Sigma-Aldrich,目录号:N1048)
  4. Tris-HCl
  5. EDTA
  6. MgCl 2
  7. 来自Birchwood的Xylan(Sigma-Aldrich,目录号:X4252)
  8. 来自柑橘属水果的果胶(Sigma-Aldrich,目录号:P9436)
  9. 磷酸钠
  10. 氢氧化钠
  11. 羟胺盐酸盐(Acros Organics,目录号:5470-11-1)
  12. 高氯酸(Sigma-Aldrich,目录号:311421-50ML)
  13. 绝对甲醇
  14. 葡萄糖五乙酸酯(Sigma-Aldrich,目录号:G2354-25G)
  15. 去离子水
  16. Bio-Rad蛋白测定(Bio-Rad Laboratories,目录号:500-0006)(可选)
  17. 提取/反应缓冲液(参见配方)
  18. 重新暂停缓冲区(请参阅配方)
  19. 酸 - 酒精溶液(参见配方)
  20. 高氯酸铁试剂(MP Biomedicals,目录号:215875)(参见配方)


  1. 锋利的剃刀刀片
  2. pH计
  3. 微量滴定板读数器
  4. 微量离心机
  5. 离心机与15 ml小瓶相容
  6. 96孔微量滴定板
  7. parafilm
  8. 锋利的剃刀刀片
  9. 10毫升注射器
  10. 轻水流实验室真空
  11. SpeedVac干衣机
  12. 涡流
  13. NanoDrop分光光度计(可选)
  14. 微量振荡器


  1. 植物材料中的脂质体蛋白提取物
    1. 收获植物的2-5克地上部分(茎,叶,长角果,花和芽),并使用锋利的刀片立即切成5毫米的段。
    2. 将植物材料放入10毫升的注射器,其尖端用Parafilm密封(图1A-B)。
    3. 加入5 ml预冷的提取/反应缓冲液。
    4. 将注射器放入离心管(尖端显示向下,图1C),并在真空器(图1D-E)中的真空处理两次,持续15分钟,在10℃的冷室中5分钟。
    5. 打开注射器的尖端,小心地通过重力流动排出缓冲液并丢弃它。
    6. 在10℃下以1,000xg离心10分钟。
    7. 在冰上在管的底部聚集脂质体提取物。
    8. 通过NanoDrop或通过在 Bradford蛋白测定( 他,2011)。

      图1. Apoplast提取工具。 初始设定:5ml注射器,15ml离心管,石蜡膜。 B.注射器尖端用石蜡膜密封。 C.将密封尖端的注射器置于离心管中。 D.具有密封尖端的注射器在离心管中放置在真空释放器中。 E.真空除尘器连接到真空管。

  2. 乙酰酯酶的总活性
    1. 用提取/反应缓冲液稀释质外体提取物,得到0.128mg(100μl) 注意:用于测定的蛋白质的量将根据提取物中存在的乙酰酯酶的酶活性和特异性而变化。在这里,我们执行拟南芥提取物。
    2. 准备空白阴性对照,通过加入1微升的200mM pNP-乙酰基到100微升含有0.128毫克蛋白质煮沸10分钟的质外体提取物。
    3. 加入1微升200毫米pNP乙酰基100微升稀释的质外体提取物。
    4. 在室温下孵育0,5,15,30,60,120,180分钟读数空白,在每个时间点在微板读数器中在460nm处的样品吸光度。
    5. 通过测量在0.1mM至2mM的系列浓度中稀释的100μlpNP溶液在460nm处的吸光度来制备标准曲线。所有稀释液以3次重复制备。计算将使STD1值等于0.1μmolpNP的吸光度的线的斜率。
    6. 对于每个时间点测量OD <460>样品-OD <460>空白并计算斜率。
    7. 使用以下公式计算释放的pNP的量:
      斜率/STD1 * 0.1/0.128
  3. 使用天然底物(细胞壁衍生的多糖)测定乙酰酯酶活性,
    1. 在提取/反应缓冲液中加入0.12 mg的apopalst蛋白质至总体积为0.6 ml的20 mg天然底物。
    2. 通过在提取/反应缓冲液中向20mg天然底物中加入0.12mg煮沸的质外体蛋白来制备空白阴性对照。
    3. 在室温下孵育0,30分钟,1小时,3小时,6小时以130rpm振荡。
    4. 在每个时间点离心多糖材料(在17,000×g下1分钟),并取100μl上清液。
    5. 使用SpeedVac干燥等分试样。
    6. 将干物质重新悬浮于40μlRe-悬浮缓冲液中(使用涡旋)。
    7. 加入100μl去离子水,小心混合。
    8. 加入100微升酸 - 酒精溶液,仔细混匀。
    9. 加入260微升高氯酸铁试剂,仔细混合。
    10. 在室温下孵育15-30分钟。
    11. 测量510 nm处的吸光度。
    12. 对于每个时间点测量OD <510>样品-OD <510>空白,并计算斜率。
    13. 使用葡萄糖五乙酸酯制备标准曲线,在40μl溶液中进行从0.1mM至2mM的一系列稀释,接着从上面C7-12段。 计算斜率,其将给出STD2值等于0.2nmoles乙酰基残基的吸光度。
    14. 通过以下公式计算从天然底物释放的乙酰基的量:
      斜率* 0.2/STD2/0.02
      0.02(总共6次测量中使用0.18mg的质外体蛋白) 确定的值将表示每1分钟每1mg质外体蛋白质释放的乙酰基的量(nmoles)


  1. 提取缓冲区
    25mM Tris-HCl
    50mM EDTA
    150mM MgCl 2(pH 7.4)
  2. 重新暂停缓冲区
    0.25M羟胺盐酸盐 1M氢氧化钠溶液
  3. 酸 - 酒精溶液
  4. 高氯酸铁试剂


作者承认Carver Funding支持这项工作。


  1. 他,F.(2011)。 Bradford蛋白质测定生物协议 1(6):e45。
  2. McComb,E。和McCready,R。(1957)。 果胶和乙酰化碳水化合物聚合物中乙酰基的测定 Anal Chem 29(5):819-821。
  3. Pogorelko,G.,Lionetti,V.,Fursova,O.,Sundaram,R.M.,Qi,M.,Whitham,S.A。,Bogdanove,A.J.,Bellincampi,D.and Zabotina,拟南芥的和 转基因植物的 构巢曲霉乙酰酯酶具有降低的多糖乙酰化程度和增加的对 病原体。植物生理学 162(1):9-23。
  4. Pogorelko,G.,Fursova,O.,Lin,M.,Pyle,E.,Jass,J.and Zabotina,O.A。(2011)。 通过在质外体中表达微生物水解酶来合成修饰植物细胞壁。 Plant Mol Biol 77(4-5):433-445。 
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Pogorelko, G. and Zabotina, O. A. (2014). Assays for Determination of Acetylesterase Activity and Specificity Using pNP-acetyl and Acetylated Polysaccharides as Substrates . Bio-protocol 4(3): e1037. DOI: 10.21769/BioProtoc.1037.
  2. Pogorelko, G., Lionetti, V., Fursova, O., Sundaram, R. M., Qi, M., Whitham, S. A., Bogdanove, A. J., Bellincampi, D. and Zabotina, O. A. (2013). Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens. Plant Physiol 162(1): 9-23.