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Product Analysis of Starch Active Enzymes by TLC
采用 TLC法对淀粉活化酶进行成品分析   

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Abstract

Thin layer chromatography (TLC) is a useful technique for detecting the presence of monosaccharides through to oligosaccharides, though it needs to be optimized for the specific sugars that are analyzed. Here we present a method for visualizing the reaction product(s) of starch active enzymes, which can contain α-1, 4 linked and α-1, 6 linked glucose. This was first published in Molecular Microbiology (Cockburn et al., 2015). The TLC protocol is an adapted version of that published by Robyt and Mukerjea (Robyt and Mukerjea, 1994). For a summary of the products generated by starch active enzymes see the review by Hii et al. (2012).

Keywords: Thin Layer Chromatography(薄层色谱法), Starch(淀粉), Amylase(淀粉酶), Maltooligosaccharides(麦芽寡糖)

Materials and Reagents

  1. TLC Silica gel 60 F254 20 x 20 cm plates (Note 1) (Merck Millipore Corporation, catalog number: 1.05715.0001 )
  2. Filter paper circles (15 cm diameter) (VWR International, catalog number: 470204-480 )
  3. MilliQ quality water (dH2O)
  4. Acetonitrile (anhydrous) (Sigma-Aldrich, catalog number: 271004 )
  5. Ethyl acetate (anhydrous) (Sigma-Aldrich, catalog number: 270989 )
  6. 2-propanol (Sigma-Aldrich, catalog number: I9516 )
  7. N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma-Aldrich, catalog number: 222488 )
  8. Methanol (anhydrous) (Sigma-Aldrich, catalog number: 322415 )
  9. Sulfuric acid (Fluka, catalog number: 84716 )
    Note: Currently, it is “ Sigma-Aldrich, catalog number: 84716”.
  10. Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number: D128 )
  11. α-D-Glucose (anhydrous) (Sigma-Aldrich, catalog number: 158968 )
  12. Maltose monohydrate (Sigma-Aldrich, catalog number: M9171 )
  13. Maltotriose (Sigma-Aldrich, catalog number: M8378 )
  14. Maltotetraose, DP4 (Sigma-Aldrich, catalog number: 47877 )
  15. Maltopentaose (TCI America, catalog number: M1023 )
  16. Maltohexaose (Sigma-Aldrich, catalog number: M9153 )
  17. Maltoheptaose (Sigma-Aldrich, catalog number: 284017 )
  18. Isomaltose (Sigma-Aldrich, catalog number: I7253 )
  19. D-Panose (Sigma-Aldrich, catalog number: P2407 )
  20. Glycogen from bovine liver (Sigma-Aldrich, catalog number: G0885 )
  21. Amylopectin from potato starch (Sigma-Aldrich, catalog number: A8515 )
  22. Amylose from potato (Sigma-Aldrich, catalog number: A0512 )
  23. Dextran from Leuconostoc spp. (Sigma-Aldrich, catalog number: 31389 )
  24. Pullulan from Aureobasidium pullulans (Sigma-Aldrich, catalog number: P4516 )
  25. Your enzyme of interest and appropriate buffer
  26. Mobile phase (see Recipes)
  27. Staining solution (see Recipes)
  28. Oligosaccharide standards (see Recipes)
  29. Glycogen solution (see Recipes)
  30. Amylopectin solution (see Recipes)
  31. Amylose solution (see Recipes)
  32. Dextran solution (see Recipes)

Equipment

Note: All equipment needed is shown in Figure 1.

  1. TLC developing chamber (VWR International, catalog number: 21432-739 )
  2. Chromatography sprayer (Note 2) (Sigma-Aldrich, catalog number: Z529729 )
  3. Oven capable of reaching 120 °C (Boekel Scientific, catalog number: 107905 )
  4. (Optional) MilwaukeeTM Dual Temperature Heat Gun (Thermo Fisher Scientific, Fisher Scientific, catalog number: 19-313-598 )
  5. CorningTM ScholarTM PC-170 Hotplate (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-641-564 )


    Figure 1.
    Equipment needed for this protocol

Procedure

Enzyme reaction

  1. Prepare 20x concentrated solutions of the enzyme of interest and the appropriate buffer. The concentration of enzyme required will be enzyme and substrate specific, but 1 µg/ml final concentration (20 µg/ml stock) is likely to be a reasonable starting place.
  2. Mix 90 µl of each substrate to be studied with 5 µl of concentrated buffer and pre-incubate at the desired temperature.
  3. Add 5 µl of the concentrated enzyme, mix by pipetting up and down and take a 20 µl sample, immediately freezing or otherwise halting the reaction. Continue to take samples at 10 min, 20 min and 30 min (Note 3), freezing the samples until you are ready to perform TLC.

TLC

  1. Add 100 ml of the mobile phase to the TLC developing chamber. Soak 1 or 2 pieces of the filter paper in the mobile phase and stick it to the wall of the chamber (see Note 4). Replace the lid of the chamber and allow 30 min for equilibration of the mobile phase with the atmosphere of the chamber.
  2. With a pencil draw a line 2 cm from the bottom of the TLC plate and include evenly spaced hashmarks (at least 1 cm apart) for each sample to be loaded. These may be labelled below the line in pencil.
  3. Load 1 µl of each standard and sample. Standards may be combined to save space, though at a slight cost to resolution. For less concentrated samples the initial spot can be allowed to dry prior to addition of another 1 µl drop in the same position. This can be done repeatedly as a method for concentrating the sample.
  4. Once the spots have dried place the TLC plate in the developing chamber, immersing the bottom of the plate into the mobile phase at the bottom. The liquid level of the mobile phase should not come above your pencil line. Replace the cover on the chamber.
  5. Incubate until the mobile phase reaches the top of the plate, approximately 3 h for a 20 cm plate.
  6. To separate longer oligosaccharides (>approximately 12 monosaccharide units), remove the plate from the chamber, allow it to dry and then put it back in the chamber to perform a second ascent.

    Video 1. Steps of TLC procedure

Staining

  1. Allow the plate to dry and then spray the plate with the staining solution, coating it evenly.
  2. Allow the plate to dry and then place it in the oven or use a heat gun at 120 °C, until spots become clearly visible, approximately 10 min in oven, somewhat less with the heat gun.

Analysis

  1. Use the standards to determine the identity of the products produced. Note that sugars containing α-1, 6 linkages will run approximately equivalent to an all α-1, 4 linked oligosaccharide one monosaccharide unit longer, for each α-1, 6 linkage present. e.g. isomaltose, runs at approximately the same distance as maltotriose and panose runs at the same distance as maltotetraose (see Figure 2).

Representative data


Figure 2. Sugar standards. Illustration of the run profile for sugar standards. Maltose through maltoheptaose are α-1, 4 linked polymers of glucose from DP2 to DP7. Isomaltose is glucose α-1, 6 linked to glucose, panose is glucose α-1, 6 linked to maltose and glucosyl maltotriose is glucose α-1, 6 linked to maltotriose. Each standard was at 5 mM and 1 µl was loaded onto the plate.

Notes

  1. Smaller format plates can be used, particularly if investigating the production of shorter oligosaccharides (maltoheptaose or smaller).
  2. In the absence of a sprayer the plate can be dipped in the staining solution or the staining solution can be poured over the plate, though streaking is more likely to occur.
  3. This procedure is designed to investigate initial reaction products. It is also useful in some cases, particularly with debranching enzymes (hydrolyzing α-1, 6 linkages), to examine the final reaction products. This can be accomplished by using a higher concentration of enzyme and/or a longer incubation time, allowing the reaction to proceed to completion.
  4. Soaking the filter paper and sticking it to the wall of the chamber is optional, but will shorten the time needed for equilibration to occur. This step is not shown in Video 1.
  5. For storage of the polysaccharide solutions pass through a 0.2 µm filter and add sodium azide to 0.02% and store at room temperature (Lichstein and Soule, 1944). For oligosaccharide solutions, store frozen.

Recipes

  1. Mobile phase
    Mix 85 ml acetonitrile, 20 ml ethyl acetate, 50 ml 2-propanol, 60 ml water
    Requires approximately 100 ml per assay (less with smaller TLC plates and chambers)
  2. Staining solution
    Add 10 ml of sulfuric acid to 190 ml of methanol
    Dissolve 0.6 g of N-(1-naphthyl)ethylenediamine dihydrochloride
  3. Oligosaccharide standards
    Make to 5 mM in water
  4. Glycogen solution
    Dissolve 150 mg in 50 ml of water
  5. Amylopectin solution
    Dissolve 150 mg in 3 ml DMSO
    Slowly add hot water (above 60 °C) with vigorous stirring, bringing the volume up to 50 ml
  6. Amylose solution
    Dissolve 75 mg in 10 ml DMSO
    Slowly add very hot water (above 80 °C) with vigorous stirring, bringing volume up to 50 ml
  7. Dextran solution
    Dissolve 150 mg in 50 ml of water

Acknowledgments

This work was supported by startup funds to Nicole Koropatkin. The TLC separation protocol was adapted from the work of Robyt and Mukerjea (1994).

References

  1. Cockburn, D. W., Orlovsky, N. I., Foley, M. H., Kwiatkowski, K. J., Bahr, C. M., Maynard, M., Demeler, B. and Koropatkin, N. M. (2015). Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale. Mol Microbiol 95(2): 209-230.
  2. Hii, S. L., Tan, J. S., Ling, T. C. and Ariff, A. B. (2012). Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res 2012: 921362.
  3. Lichstein, H. C. and Soule, M. H. (1944). Studies of the effect of sodium azide on microbic growth and respiration: I. The action of sodium azide on microbic growth. J Bacteriol 47(3): 221-230.
  4. Robyt, J. F. and Mukerjea, R. (1994). Separation and quantitative determination of nanogram quantities of maltodextrins and isomaltodextrins by thin-layer chromatography. Carbohydr Res (251) 187-202.

简介

薄层色谱(TLC)是用于检测单糖通过寡糖的存在的有用技术,尽管它需要针对所分析的特定糖进行优化。 在这里我们提出一种可视化的淀粉活性酶,可以包含α-1,4连接和α-1,6连接葡萄糖的反应产物的方法。 这首次发表于Molecular Microbiology(Cockburn等人,2015年)。 TLC方案是由Robyt和Mukerjea(Robyt和Mukerjea,1994)发表的适应版本。 对于由淀粉活性酶产生的产物的概述,参见Hii等人(2012)的综述。

关键字:薄层色谱法, 淀粉, 淀粉酶, 麦芽寡糖

材料和试剂

  1. TLC硅胶60F 254/20×20cm板(注1)(Merck Millipore Corporation,目录号:1.05715.0001)
  2. 滤纸圆(直径15cm)(VWR International,目录号:470204-480)
  3. MilliQ质量水(dH 2 O)
  4. 乙腈(无水)(Sigma-Aldrich,目录号:271004)
  5. 乙酸乙酯(无水)(Sigma-Aldrich,目录号:270989)
  6. (Sigma-Aldrich,目录号:I9516)
  7. N-(1-萘基)乙二胺二盐酸盐(Sigma-Aldrich,目录号:222488)
  8. 甲醇(无水)(Sigma-Aldrich,目录号:322415)
  9. 硫酸(Fluka,目录号:84716)
    注意:目前,它是"Sigma-Aldrich,目录号:84716"。
  10. 二甲基亚砜(DMSO)(Thermo Fisher Scientific,目录号:D128)
  11. α-D-葡萄糖(无水)(Sigma-Aldrich,目录号:158968)
  12. 麦芽糖一水合物(Sigma-Aldrich,目录号:M9171)
  13. 麦芽三糖(Sigma-Aldrich,目录号:M8378)
  14. 麦芽四糖,DP4(Sigma-Aldrich,目录号:47877)
  15. 麦芽五糖(TCI America,目录号:M1023)
  16. 麦芽六糖(Sigma-Aldrich,目录号:M9153)
  17. 麦芽七糖(Sigma-Aldrich,目录号:284017)
  18. 异麦芽糖(Sigma-Aldrich,目录号:I7253)
  19. D-Panose(Sigma-Aldrich,目录号:P2407)
  20. 来自牛肝的糖原(Sigma-Aldrich,目录号:G0885)
  21. 来自马铃薯淀粉(Sigma-Aldrich,目录号:A8515)的支链淀粉
  22. 来自马铃薯的直链淀粉(Sigma-Aldrich,目录号:A0512)
  23. 来自 Leuconostoc 的葡聚糖。 (Sigma-Aldrich,目录号:31389)
  24. 来自洋葱芽孢杆菌的支链淀粉(Sigma-Aldrich,目录号:P4516)
  25. 您感兴趣的酶和适当的缓冲液
  26. 流动相(参见食谱)
  27. 染色溶液(见配方)
  28. 低聚糖标准(参见配方)
  29. 糖原溶液(见配方)
  30. 支链淀粉溶液(参见配方)
  31. 直链淀粉溶液(参见配方)
  32. 葡聚糖溶液(见配方)

设备

注意:图1中显示了所有需要的设备。

  1. TLC显影室(VWR International,目录号:21432-739)
  2. 色谱喷雾器(注2)(Sigma-Aldrich,目录号:Z529729)
  3. 能够达到120℃的烘箱(Boekel Scientific,目录号:107905)

  4. Milwaukee TM双温度热枪(Thermo Fisher Scientific,Fisher Scientific,目录号:19-313-598)
  5. Corning TM Scholar PC-170热板(Thermo Fisher Scientific,Fisher Scientific,目录号:13-641-564)


    图1. 此协议所需的设备

程序

酶反应

  1. 准备20倍浓缩的目标酶的溶液和适当的缓冲液。所需酶的浓度将是酶和底物特异性,但是1μg/ml终浓度(20μg/ml储备液)可能是合理的起始位置。
  2. 混合90微升待研究的每种底物与5微升浓缩缓冲液和预孵育在所需的温度
  3. 加入5μl浓缩酶,通过吸取上下混合,取20μl样品,立即冻结或停止反应。继续在10分钟,20分钟和30分钟取样(注3),冷冻样品,直到您准备好进行TLC。

TLC

  1. 加入100ml的流动相到TLC显影室。在流动相中浸泡1或2块滤纸,并将其粘在腔室壁上(见注4)。更换腔室的盖子,并允许流动相与腔室的气氛平衡30分钟
  2. 用铅笔从TLC板的底部画2厘米的线,并为每个要装载的样品包括均匀间隔的标记(至少1厘米)。这些可以在铅笔下方的线下标记。
  3. 加载1μl的每个标准品和样品。标准可以组合以节省空间,虽然以微小的成本来解决。对于较低浓度的样品,可以使初始点干燥,然后在相同位置加入另外1μl液滴。这可以作为浓缩样品的方法重复进行。
  4. 一旦斑点干燥,将TLC板放在显影室中,将板的底部浸入底部的流动相中。流动相的液位不应超过铅笔线。更换腔室上的盖子。
  5. 孵育直到流动相到达板的顶部,对于20cm板大约3小时。
  6. 为了分离较长的寡糖(>大约12个单糖单元),将板从室中取出,使其干燥,然后将其放回室中以进行第二次上升。

    视频1. TLC过程的步骤
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染色

  1. 使板干燥,然后用染色溶液喷涂板,均匀涂布
  2. 让板干燥,然后放在烤箱或使用热风枪在120°C,直到斑点变得清晰可见,约10分钟在烤箱,稍微少一些热风枪。

分析

  1. 使用标准来确定所生产产品的身份。注意,对于存在的每个α-1,6键,含有α-1,6键的糖将大约等于所有α-1,4连接的寡糖一个单糖单元更长。例如异麦芽糖,以与麦芽三糖大致相同的距离运行,并且panose以与麦芽四糖相同的距离运行(参见图2)。

代表数据


图2.糖标准。糖标准的运行配置文件示例。麦芽糖通过麦芽糖是从DP2到DP7的葡萄糖的α-1,4连接聚合物。异麦芽糖是与葡萄糖连接的葡萄糖α-1,6,与葡萄糖连接的葡萄糖α-1,6,与麦芽糖连接的葡萄糖α-1,6,葡萄糖麦芽三糖是与麦芽三糖连接的葡萄糖α-1,6。每个标准品为5mM,并将1μl加载到平板上。

笔记

  1. 可以使用更小的格式板,特别是如果研究较短的寡糖(麦芽七糖或更小)的生产。
  2. 在没有喷雾器的情况下,可以将板浸入染色溶液中,或者可以将染色溶液倒在板上,尽管更可能发生条纹。
  3. 该程序设计用于研究初始反应产物。它在一些情况下也是有用的,特别是对于脱支酶(水解α-1,6键),以检查最终反应产物。这可以通过使用更高浓度的酶和/或更长的温育时间来完成,允许反应进行至完成。
  4. 浸泡滤纸并将其粘附在室壁上是可选的,但会缩短平衡发生所需的时间。此步骤不会显示在视频1中。
  5. 为了储存多糖溶液,通过0.2μm过滤器并加入叠氮化钠至0.02%并在室温下储存(Lichstein和Soule,1944)。对于寡糖溶液,冷冻保存。

食谱

  1. 流动相
    混合85ml乙腈,20ml乙酸乙酯,50ml 2-丙醇,60ml水
    每次测定需要约100 ml(更小的TLC板和小室)
  2. 染色溶液
    将10ml硫酸加入到190ml甲醇中 溶解0.6g N-(1-萘基)乙二胺二盐酸盐
  3. 低聚糖标准品
    制成5mM水溶液
  4. 糖原溶液
    将150mg溶于50ml水中
  5. 支链淀粉溶液
    将150mg溶于3ml DMSO中 在剧烈搅拌下缓慢加入热水(60°C以上),使体积达到50ml
  6. 直链淀粉溶液
    将75mg溶于10ml DMSO中 在剧烈搅拌下缓慢加入非常热的水(80°C以上),使体积达到50ml ml
  7. 葡聚糖溶液
    溶于150毫升水中

致谢

这项工作是由创业基金支持Nicole Koropatkin。 TLC分离方案改编自Robyt和Mukerjea(1994)的工作。

参考文献

  1. Cockburn,D.W.,Orlovsky,N.I.,Foley,M.H.,Kwiatkowski,K.J.,Bahr,C.M.,Maynard,M.,Demeler,B.and Koropatkin,N.M。 人类肠道共生菌中的淀粉利用途径的分子细节直肠真实杆菌 。 Mol Microbiol 95(2):209-230。
  2. Hii,S.L.,Tan,J.S.,Ling,T.C.and Ariff,A.B。(2012)。 支链淀粉酶:淀粉水解和潜在工业应用中的作用 Enzyme Res 2012:921362.
  3. Lichstein,H.C.and Soule,M.H。(1944)。 叠氮钠对微生物生长和呼吸的影响研究:I.叠氮钠对微生物生长。细菌 47(3):221-230
  4. Robyt,J.F。和Mukerjea,R。(1994)。 通过薄层色谱法分离和定量测定纳克量的麦芽糖糊精和异麦芽糖糊精。 Carbohydr Res (251)187-202。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cockburn, D. and Koropatkin, N. (2015). Product Analysis of Starch Active Enzymes by TLC. Bio-protocol 5(20): e1621. DOI: 10.21769/BioProtoc.1621.
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