Name: automated modular high throughput exopolysaccharide screening platform coupled with highly sensitive carbohydrate fingerprint analysis
Uploaded: 2016-04-11T13:00:00.000+00:00
Duration: 12 min 2 s
Description: Watch this Scientific Journal Video about Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis at JoVE.com

We sincerely thank Thomas Howe and J&#246;rg Carsten for the programming and technical support with the liquid handling systems.

<ol>
	<li>Milani, J., Maleki, G., Valdez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2,+Hydrocolloids+in+Food+Industry.">Ch. 2, Hydrocolloids in Food Industry.</a> Food Industrial Processes - Methods and Equipment. 2, (2012).</li><li>Thibodeau, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+the+skin+from+environmental+stresses+with+an+exopolysaccharide+formulation.">Protecting the skin from environmental stresses with an exopolysaccharide formulation.</a> Cosmet Toiletries. 120 (12), 81-90 (2005).</li><li>Prajapati, V. D., Jani, G. K., Zala, B. S., Khutliwala, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+the+emerging+exopolysaccharide+gellan+gum+as+a+novel+polymer.">An insight into the emerging exopolysaccharide gellan gum as a novel polymer.</a> Carbohydr Polym. 93 (2), 670-678 (2013).</li><li>Schmidt, W., Brouwers, H. J. H., Kuehne, H. C., Meng, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+working+mechanism+of+starch+and+diutan+gum+in+cementitious+and+limestone+dispersions+in+presence+of+polycarboxylate+ether+superplasticizers.">The working mechanism of starch and diutan gum in cementitious and limestone dispersions in presence of polycarboxylate ether superplasticizers.</a> Appl. Rheol. 23 (5), 52903 (2013).</li><li>Srinivasan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+Polysaccharides+as+Treatment+Agents+for+Wastewater.">Natural Polysaccharides as Treatment Agents for Wastewater.</a> Green Materials for Sustainable Water Remediation and Treatment. , 51-81 (2013).</li><li>R&#252;hmann, B., Schmid, J., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+exopolysaccharide+screening+platform:+From+strain+cultivation+to+monosaccharide+composition+and+carbohydrate+fingerprinting+in+one+day.">High throughput exopolysaccharide screening platform: From strain cultivation to monosaccharide composition and carbohydrate fingerprinting in one day.</a> Carbohydr Polym. 122, 212-220 (2015).</li><li>Ortega-Morales, B. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+extracellular+polymers+synthesized+by+tropical+intertidal+biofilm+bacteria.">Characterization of extracellular polymers synthesized by tropical intertidal biofilm bacteria.</a> J Appl Microbiol. 102 (1), 254-264 (2007).</li><li>Xu, R., Ma, S., Wang, Y., Liu, L., Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+identification+and+statistic+optimization+of+a+novel+exopolysaccharide+producing+Lactobacillus+paracasei+HCT.">Screening identification and statistic optimization of a novel exopolysaccharide producing Lactobacillus paracasei HCT.</a> Afr. J. Microbiol. Res. 4 (9), 783-795 (2010).</li><li>Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+method+for+determination+of+sugars+and+related+substances.">Colorimetric method for determination of sugars and related substances.</a> Anal. Chem. 28 (3), 350-356 (1956).</li><li>Masuko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+analysis+by+a+phenol-sulfuric+acid+method+in+microplate+format.">Carbohydrate analysis by a phenol-sulfuric acid method in microplate format.</a> Anal Biochem. 339 (1), 69-72 (2005).</li><li>Geater, C. W., Fehr, W. R., Wilson, L. A., Robyt, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+more+rapid+method+of+total+sugar+analysis+for+soybean+seed.">A more rapid method of total sugar analysis for soybean seed.</a> Crop Sci. 41 (1), 250-252 (2001).</li><li>Sutherland, I. W., Dumitriu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+16,+Microbial+Exopolysaccharides.">Ch 16, Microbial Exopolysaccharides.</a> Polysaccharides Structural Diversity and Functional Versatility. , 431-457 (2005).</li><li>Wingender, J., Neu, T. R., Flemming, H. C., Wingender, J., Neu, T. R., Flemming, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+Bacterial+Extracellular+Polymeric+Substances?.">What are Bacterial Extracellular Polymeric Substances?.</a> Microbial extracellular polymeric substances: characterization, structure and function. , 258 (1999).</li><li>R&#252;hmann, B., Schmid, J., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+carbohydrate+analysis+via+liquid+chromatography+coupled+with+ultra+violet+and+electrospray+ionization+ion+trap+detection+in+96-well+format.">Fast carbohydrate analysis via liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection in 96-well format.</a> J. Chromatogr. A. 1350, 44-50 (2014).</li></ol>

We have nothing to disclose.

多糖检测与酚-硫酸-硫酸法:不同的单糖通过展示使用这种方法9不同的最大吸收和摩尔消光系数。这导致聚合物，其含有不同量的几种糖不同的吸收最大值。吸收最大值的16不同市售聚合物不同波长在表5中给出。在聚合物中溶解（1克/升）在DDH 2 O中，搅拌（150rpm）下过夜，并用苯酚-硫酸-酸法测定。的diutan胶表现出最低的最大吸收在470nm和硬葡聚糖和透明质酸最高在484纳米。基于这些结果被选择为480nm此筛选平台。的聚合物的相对吸光度是根据用1克/升葡萄糖（设定为100％）得到的吸光度计算出来的。最低的结果，获得与核菌葡聚糖和琥珀，都与92％。这是EXPected因为硬葡聚糖只含有葡萄糖和琥珀酰聚糖含有以7:3的比例的葡萄糖和半乳糖:1。商用聚合物具有干燥和不同灰分含量的不同的损失，这就是为什么没有达到〜110％的理论值的原因。木聚糖显示出与311％的最高相对吸光度。这样做的原因是，从木糖达到的高摩尔消光系数，由于更占优势呋喃糖形式。以0.1克/升葡萄糖的水平的定量极限为止，以及在一个浓度&lt;0.05克/升的检测极限。然而，对于在筛选阳性菌株的检测极限是更高大于0.7克/升，因此，该试验显示出良好的性能。为了得到可靠的结果，凝胶过滤后剩余的葡萄糖与葡萄糖的测定法来确定，该值是从由苯酚 - 硫酸 - 酸法的值中减去。 自动葡萄糖测定稀释:性能栽培（稀释1:100）之后的葡萄糖测定进行了研究。对于这一点，将10μl的上清液在深孔板经由十倍抽吸和分配180微升出该稀释的转移到990微升DDH 2 O的和混合。第二个关键步骤是只有5从凝胶过滤后的葡萄糖测定中1:10稀释微升等分正确的移液。为了产生稀释25μl的DDH 2 O的用50微升尖首先被转移，之后20微升的DDH 2 O和5μl的凝胶滤液被一起吸出。这确保了尖端的更好除去5μl的等分试样。这两个稀释步骤通过葡萄糖测定各种葡萄糖标准进行了核实。对于两个示例性浓度，结果在表6中给出的1:100稀释的葡萄糖含量的测定后培养表明高精度用于与CV＆＃两种标准60; 1.2％。同时，对于更高的标准精度高达11.6（％误差）。然而，这是可忽略不计的葡萄糖测定仅代表培养后剩余的葡萄糖含量，因此，是不为聚合物检测重要。在1:10稀释为凝胶过滤后剩余的葡萄糖表现出非常可靠的结果与CV &lt;1.4％和精确度&lt;2.1％的误差。 考虑蒸发的:筛选需要从第一步骤到第一葡萄糖的测定3.5小时。为了找到答案，这个时间段是否有不封顶MTP存储的影响，50微升的葡萄糖检测校准标准进行储存，没有遮挡机器人传送带3.5小时。在校准范围（45到4.5毫克/升）的样品浓度几乎不增加。增加 - 引起的蒸发 - 低于2.1％和仅两个最低浓度（1.8和0.9毫克/升）则达到高达12.4％（ <str翁&gt;表7）。 凝胶过滤:高含量的非代谢葡萄糖的血糖扰乱从水解聚合物的定量检测。因此，需要凝胶过滤步骤培养后，除去剩余的葡萄糖。此外，凝胶过滤净化含有盐和单体碳水化合物的化合物，更为比葡萄糖上清液中的聚合物，以减少在该单体的分析的分析的背景。在凝胶过滤步骤35微升滤液置于井的中心。用于凝胶过滤的自动化系统的稳健性的验证，从0.045 8校准标准高达9克/升葡萄糖被过滤（N = 8）。每一浓度的葡萄糖总是由初始值（ 表8）的95％以上的降低。在这样做时，凝胶过滤显示关于各种浓度的葡萄糖的非常好的结果。另外，剩余的葡萄糖后凝胶-Filtration也与一个葡萄糖的测定法测定，并从苯酚 - 硫酸 - 酸判定减去接收葡萄糖当量的正确量的水解聚合物。 丙酮酸的测定:首先，研究了在中和和稀释（1:10）从水解步骤TFA-矩阵是否与酶反应的干扰。因此，进行了完整的测定两次，一次用和一次性无基质和显示可靠的结果。最后，16市售聚合物的丙酮酸含量成功测量并在图3中示出，它通常已知掉那些16的聚合物的仅琥珀酰聚糖和黄原天然含有丙酮酸盐。随着我们的丙酮酸法这两种聚合物被正确识别。在硬葡聚糖，在显著量也检测Welan胶和木聚糖丙酮酸盐。总体而言，该方法的能力进行了验证和丙酮酸的测定小号howed高性能。它被证明是能够水解后，以检测在不同的聚合物丙酮酸盐。 碳水化合物指纹:执行中的自动筛查所有分析模块后，被选定为碳水化合物指纹图谱分析潜在的EPS生产商。对于这一点，被应用的几个标准:1）粘度的正观测离心后和/或之后过滤。 2）沉淀过滤前后。观察纤维薄片被评价为阳性。 3）从苯酚 - 硫酸 - 酸法葡萄糖等效值。值&gt; 700毫克/升被评为积极，300和700毫克/升之间的值被评为公认的EPS生产商。当两个或三个标准被作为阳性评价，被选择用于进一步的碳水化合物指纹分析的菌株。该标准可以对EPS筛查（ 如低粘度EPS）的个人目的进行定制。我们的方法旨在寻找effici耳鼻喉科EPS生产商。当菌株只产生少量的EPS的搜索葡萄糖当量的评价极限应减少。 技术效益和未来的应用:该协议的一个有趣的特点是的步骤和不同的分析模块，模块化的特点。它们可以以不同的方式进行组合，调整至个体需要和新颖的模块可以容易地实现。此外，分析模块可以单独使用， 例如 ，在组合的水解模块对HT-PMP-衍生模块能够从在96-孔格式不同的聚合物溶液（1克/升）进行单体组合物的分析。为无需访问液体处理系统的完整筛选可以手动处理而不会在移液方案的任何改变的实验室。然而，使用液体处理系统增加了吞吐量高达768株（而不是192株如果screene手动D）每天。此处描述的协议能够为不同属的筛选，因此，对于大应变集合，以确定新的车生产者和分析其碳水化合物指纹在一个方法（ 图4）的筛选。此外，对于含有稀土糖，如岩藻糖，糖醛酸，甚至未知糖多糖靶向筛选可通过详细单糖分析进行。另外，在定义的比率不同的糖组合可以被检测出来。这使得已知的EPS或EPS新颖的结构相关的变种简单的识别。

微生物胞外多糖（EPS）是满足各种生物功能聚合物的结构高度多样化的群体。它们通常都建复杂的重复单元，其由不同类型的单体（糖，糖的衍生物，糖酸），这些单体和它们的取代之间的键区分开的。微生物多糖的结构多样性赋予这种分子类，允许类的食物1不同的领域及其应用，化妆品2,3，建筑化学4或水处理5的成员，而不同的特点。以进一步扩展这些生物基和这种可持续聚合物新颖天然微生物多糖的识别以及结构变体的工程表示有前途的方法的应用领域。无论哪种方式，需要快速筛选方法来快速扫描兵卫微生物菌种的广大- [R多糖的形成，并分析他们的产品。因此，我们最近开发了用于从天然分离或工程菌株的变体，其包括单体组合物6的识别微生物多糖生产分析的96孔HT-筛选平台。 应用此平台的第一轮筛选〜500天然分离的使我们能够确定只有约10％至20％的分离株为能够产生车（未示出数据）。这意味着，所分析的菌株的80〜90％没有产生所施加的条件下的EPS，因此，详细的碳水化合物指纹的一个进一步的分析是没有必要的。作为这种高度复杂的识别单体组合物是一个耗时的过程，特别是对于数据分析，快速的预筛选方法，以确定该菌株的EPS生产正，会急剧增加的效率。此外，试剂耗材和测量时间的UHPLC-UV-ESI-MS将会减少。此外，不同的分析模块，虽然，一方面使该方法高度可靠的，是在另一方面复杂多于两个96孔板的并联的手工处理，因此限制了该方法的全部潜力。由于这些原因，我们决定开发一个自动化筛选平台。因此，我们结合总糖含量的完全自动化的快速检测方法的现有碳水化合物指纹技术的模块化格式，根据吸光度测定。 酚-硫酸-酸方法仍然代表所选择的用于快速确定细菌和植物多糖7,8的总碳水化合物含量的方法。这种方法首先由Dubois 等人 9在96-孔板10,11描述的和适于不同的应用和样本大小，即使是小规模的。苯丙氨酸NOL - 硫酸酸方法测量一个值的总碳水化合物的含量，所有求和单体，低聚物和样品的聚合碳水化合物。 考虑到这些方面考虑，合适的培养介质的选择是必不可少的应用此方法。含低聚或类似酵母提取物高分子糖类化合物复杂的介质可能导致改变聚合物的含量，因此，应严格避免。此外，大量的糖被用作C-源菌株的培养。从栽培过程中其余的碳水化合物高水平可能负面与EPS内容的干扰测量。 因此，使用定义和纯糖的建议。在我们的实验中，我们使用葡萄糖的细胞的培养。培养后剩余的葡萄糖溶液通过凝胶过滤减少和通过葡萄糖的测定法来确定。最后，葡萄糖当量多糖第被已经用苯酚 - 硫酸 - 酸法检测到的从总碳水化合物含量凝胶过滤后减去剩余的葡萄糖计算。凝胶过滤和葡萄糖的测定与苯酚 - 硫酸 - 酸法组合保证可靠的结果，并且能够被提供了第一，完全自动化的检测系统。 两个新的分析模块，包括在自动化筛选平台，以增加从所述EPS检测系统的信息量:沉淀和粘度增大的观察。 许多不同的EPS - 例如琥珀酰聚糖，黄原胶和结肠酸12 -据报道，与糖位置C4和C6的非碳水化合物丙酮酸缩酮进行修改。那些丙酮酸缩酮（正如琥珀半酯和糖醛酸）向聚阴离子性质，因此，在物理prope通过经由二价阳离子桥13相互作用的聚合物的rties。为了确定这些特定聚合物丙酮酸的确定建立了作为另一其他分析模块。这增加了对多糖的取代基和其潜在的宏观性质的信息。 结合所有的模块使不同EPS的标识以及一个快速，高效的决心EPS生产商。通过这种方法的筛选平台可分为两个主要部分（ 图1）。在自动筛选（第一部分）的工作流中出现全自动化（ 表1）快速预选产生菌的EPS。碳水化合物指纹分析（第二部分）定量确定由预选菌株产生的EPS的单体组合物。从而，数据分析以优化的大应变集合的筛选最小化。这个提供分析在一个单一的自动筛选运行，并以二奔跑的每每天768株天是可能的384株的可能性。此外，该碳水化合物指纹分析给出了所有所识别的车的一个更详细的概述。这使定向分析和仅稍微不同的EPS的变体或完全新的识别相比EPS的已经描述的化学结构。

<table><tbody><tr><td>96 well deep well plate 2.0 ml (DWP)</td><td>Greiner Bio-One</td><td>780271</td><td>Main-culture (CP 1-1 to 1-4), equilibration plates for gel-filtration (CP 1-6, 1-7, 2-6 and 2-7), 1:100 dilution for the glucose assay (CP 4-1 to 4-4) containing 990&amp;nbsp;&amp;micro;l of ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Breathable Sealing Film</td><td>Axygen</td><td>BF-400-S</td><td>Incubation film for pre- and main-culture DWP</td></tr><tr><td>Aluminum Sealing Film</td><td>Axygen</td><td>PCR-AS-200</td><td>-80 &amp;deg;C storage film for glycerol-stock plates</td></tr><tr><td>MCA96 Nested Disposable Tips 200 &amp;micro;l</td><td>TECAN</td><td>30038619</td><td>Worktable position (WTP 2-1 to 2-4)</td></tr><tr><td>A/B glass-filter-plate 1&amp;nbsp;&amp;micro;m</td><td>Pall Corporation</td><td>PN 8031</td><td>Stored on collector plate (CP 2-1 to 2-4)</td></tr><tr><td>96-well micro titer plate V-Bottom</td><td>Greiner Bio-One</td><td>651201</td><td>Collector plate for filtration plate (CP 2-1 to 2-4)</td></tr><tr><td>96-well SpinColumn G-25</td><td>Harvard Apparatus</td><td>74-5612</td><td>Stored on washing DWP (CP 1-6, 1-7, 2-6 and 2-7)</td></tr><tr><td>96-well micro titer plate V-Bottom</td><td>Nunc</td><td>249944</td><td>Collector plate for gel-filtration plate (CP 3-1 to 3-4)</td></tr><tr><td>Nested Disposable Tips SBS 50 &amp;micro;l tips</td><td>TECAN</td><td>30038609</td><td>Carousel position (CP 4-6, 4-7 and 5-1 to 5-6)</td></tr><tr><td>Trough 250 ml</td><td>Axygen</td><td>Res-SW96-HP</td><td>Water WTP 1-1, Glucose assay reagent-mix WPT 1-2, ammonium-acetate buffer pH 5.6 (CP 5-7)</td></tr><tr><td>96-well micro titer plate F-Bottom (MTP)</td><td>Greiner Bio-One</td><td>655101</td><td>precipitation 1 (CP 6-1 to 6-4), pH-value (CP 7-1 to 7-4), precipitation 2 (CP 8-1 to 8-4), phenol-sulfuric-acid method (CP 8-5 to 8-8), glucose-assay (CP 9-1 to 9-9), dummy plates (WTP 3-1, 3-2)</td></tr><tr><td>96-well silicon cap mat&amp;nbsp;</td><td>Whatmann</td><td>7704-0105</td><td>Cover mat for MTP</td></tr><tr><td>200 &amp;micro;l pipette tips</td><td>Sarstedt</td><td>70.760.002</td><td>For manually handling</td></tr><tr><td>1,000 &amp;micro;l pipette tips</td><td>Sarstedt</td><td>70.762</td><td>For manually handling</td></tr><tr><td>96-well-PCR micro titer plate&amp;nbsp;</td><td>Brand</td><td>781350</td><td>Hydrolysis, PMP-derivatisation</td></tr><tr><td>TPE (thermoplastic elastomer) cap mat&amp;nbsp;</td><td>Brand</td><td>781405</td><td>Hydrolysis, PMP-derivatisation</td></tr><tr><td>Filter plate 0.2&amp;nbsp;&amp;micro;m Supor</td><td>Pall Corporation</td><td>PN 8019</td><td>Filtration of samples for UHPLC-ESI-MS analysis with a MTP collector plate</td></tr><tr><td>Pipette Tips LHS 5-300&amp;nbsp;&amp;micro;l</td><td>Brand</td><td>732150</td><td>Brand LHS system</td></tr><tr><td>Glucose oxidase&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G2133</td><td>Glucose-assay</td></tr><tr><td>Horseradish peroxidase&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P6782</td><td>Glucose-assay, pyruvat-assay</td></tr><tr><td>DA-64 (&lt;em&gt;N&lt;/em&gt;-(Carboxymethylaminocarbonyl)-4.4&#039;-bis(dimethylamino)-diphenylamine sodium salt)</td><td>Wako Chemicals GmbH</td><td>043-22351</td><td>Pyruvat-assay</td></tr><tr><td>Pyruvate oxidase&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P4591</td><td>Pyruvat-assay</td></tr><tr><td>Potassium phosphate dibasic</td><td>Carl-Roth</td><td>P749.3</td><td>Pyruvat- and glucose-assay</td></tr><tr><td>Potassium phosphate monobasic</td><td>Carl-Roth</td><td>3904.3</td><td>Pyruvat- and glucose-assay</td></tr><tr><td>Thiamine pyrophosphate</td><td>Sigma-Aldrich</td><td>C8754</td><td>Pyruvat-assay</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-Aldrich</td><td>31413</td><td>Pyruvat-assay</td></tr><tr><td>2-Propanol</td><td>VWR</td><td>20922.466</td><td>Precipitation</td></tr><tr><td>Phenol</td><td>VWR</td><td>20599.231</td><td>Phenol-sulfuric-acid method</td></tr><tr><td>Sulfuric acid</td><td>Carl-Roth</td><td>4623.4</td><td>Phenol-sulfuric-acid method</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T6508</td><td>Hydrolysis</td></tr><tr><td>Ammonium solution</td><td>Carl-Roth</td><td>P093.1</td><td>Hydrolysis, PMP-derivatization</td></tr><tr><td>Ethanol absolut</td><td>VWR</td><td>20821.321</td><td>Hydrolysis, PMP-derivatization</td></tr><tr><td>Phenol red&amp;nbsp;</td><td>Alfa Aesar</td><td>B21710</td><td>Hydrolysis, PMP-derivatization</td></tr><tr><td>1-Phenyl-3-methyl-5-pyrazolone</td><td>Sigma-Aldrich</td><td>M70800</td><td>PMP-derivatization</td></tr><tr><td>Methanol LC-MS</td><td>VWR</td><td>83638.320</td><td>PMP-derivatization</td></tr><tr><td>Acetonitril LC-MS</td><td>VWR</td><td>83040.320</td><td>PMP-derivatization</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>338826</td><td>PMP-derivatization</td></tr><tr><td>Ethanol absolut</td><td>VWR</td><td>20821.321</td><td>PMP-derivatization</td></tr><tr><td>Methyl red</td><td>Alfa Aesar</td><td>36667</td><td>pH-value</td></tr><tr><td>Robotic liquid handling system&amp;nbsp;</td><td>Tecan</td><td>Freedom EVO</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Liquid handling station LHS</td><td>Brand</td><td>709400</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Tip-Adapter</td><td>Brand</td><td>709434</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Liquid Ends MC 20-300&amp;nbsp;&amp;micro;l</td><td>Brand</td><td>709416</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Adapter 60 mm</td><td>Brand</td><td>709430</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr></tbody></table>

automated modular high throughput exopolysaccharide screening platform coupled with highly sensitive carbohydrate fingerprint analysis

许多微生物能够产生和分泌的胞外多糖（EPS），其具有在医学领域，食品应用或在更换的基于石油化工的重要影响。我们描述了一种液体处理系统，其允许的类型和EPS的由微生物产生的量的快速和可靠的分析，以进行自动化分析平台。它使用户能够确定新的天然微生物胞外多糖生产者和分析在一天之内的相应聚合物的碳水化合物指纹在高吞吐量（HT）。使用此平台，应变集合以及可能在工程方法来获得的菌株的变体的文库可以筛选。该平台有一个模块化设置，其允许分离协议的为两个主要部分。首先，有一种自动筛选系统，它结合了不同的多糖的检测模块:viscosit的半定量分析通过离心步骤，通过乙醇沉淀的聚合物的形成进行分析，并通过苯酚 - 硫酸 - 酸转化的总碳水化合物含量的测定ý形成。这里，有可能筛选高达每运行384菌株。第二部分通过组合两个基本模块为所有在第一部分中确定的选定车生产者详细单糖分析:完整单体组合物通过超高效液相色谱加上紫外线和电喷雾电离离子阱检测（分析UHPLC-UV-ESI-MS）和丙酮酸的确定为耦合到一个颜色形成通过酶促氧化的聚合物取代基（丙酮酸缩酮的存在下）。所有这种筛选平台的分析模块可以以不同的方式进行组合，并调节至个别的要求。此外，它们都可以被人工处理或与液体处理系统进行的。由此，筛选PLATForm的实现了巨大的灵活性，以确定不同的EPS。

Watch this Scientific Journal Video about Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis at JoVE.com

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

automated-modular-high-throughput-exopolysaccharide-screening

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Biochemistry of the Cell

SCIENTISTS IN ACTION

11.4K 次观看.  Technische Universität München. We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

视频: Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

总木质素，木质素单体，和酶促糖推出:顽拗变奏曲在木质纤维素生物质的高通量筛选

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

环境科学

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

生物工程

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

定量糖组学和蛋白质组学组合式净化策略

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

化学

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

生物技术环境下的氟黏膜化人乳三糖--用基因编码生物传感器分析

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

生物学

<meta charset="utf8"></head><body>使用单吸头中的微珠阵列 （BIST） 进行基于凝集素的聚糖自动化分析

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of &lt;10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

<meta charset="utf8"></head><body>作者聚焦:使用全自动系统推进蛋白质糖基化研究

A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

使用全自动系统对重组蛋白进行快速糖质定性评估

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

摘要

探索更多视频

此视频中的章节

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

总木质素，木质素单体，和酶促糖推出:顽拗变奏曲在木质纤维素生物质的高通量筛选

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

定量糖组学和蛋白质组学组合式净化策略

生物技术环境下的氟黏膜化人乳三糖--用基因编码生物传感器分析

胞外多糖的OLIGO质谱分析（OLIMP）

使用全自动系统对重组蛋白进行快速糖质定性评估

使用全自动系统对重组蛋白进行快速糖质定性评估

使用全自动系统对重组蛋白进行快速糖质定性评估

木质纤维素材料的高通量检测糖化