资助这项工作的各个方面是由各种来源，包括农业部美国农业部的具体合作协议58-6408-1-595和美国国家科学基金会（奖1049911）提供。

胞外酶活性土壤和沉积物中的比色分析 1。对酶活性的比色分析底物的制备及缓冲溶液<ol><li>通过混合50毫升0.1M乙酸（2.87毫升冰醋酸在500毫升水中）制备50mM醋酸缓冲液（pH 5.0-5.5），加入150ml的0.1M醋酸钠，和200毫升蒸馏水的H 2 O。如有必要，调整pH值至5.0-5.5用0.1M乙酸。 </li><li>在蒸馏H 2 O制备的1M氢氧化钠（NaOH）的溶液</li><li>在50mM醋酸缓冲液制备p NP-连接的底物溶液。为了测定磷酸酶制备5mM对NP-磷酸盐，在50mM乙酸盐缓冲液;以测定β-葡糖苷酶制备5mM对NP-β-吡喃葡糖苷;以测定NAG酶制备2毫米的pNP-β-N-acetylglucosaminide。准备在无菌的15毫升或50ml离心管中所有衬底的解决方案。解决方案可以被存储在4℃下2-3周。 </li></ol> 2。测定标准的转换吸光度为 p的NP浓度<ol><li>在50mM醋酸缓冲液制备的对硝基苯酚的标准溶液。浓度范围应为0.025-1毫米。 </li><li>转移100μl的各浓度重复三次，以清晰的96孔微板。加10μl1 M氢氧化钠和190微升蒸馏水H 2 O至每口井。 </li><li>在410nm下用酶标仪记录吸光度。 </li><li>乘法浓度在标准曲线0.3得到每300微升反应体积微摩尔p NP。绘制吸光度曲线与p NP的微摩尔。该曲线的斜率作为转换系数（C），这将涉及到的吸光度在各酶反应微摩尔p NP的。 </li></ol> 3。进行酶分析<ol><li>对于土壤，制备各样品的浆液在concentr待测定在无菌15ml离心管振动性为1g /毫升-1用50mM醋酸缓冲液约的。对于沉积物，补充足够的醋酸盐缓冲液，使料浆容易pipettable。所需浆料的​​确切量将根据酶测定的数目而变化，但至少为5毫升的建议。每个涡浆，直到土壤或沉积物的所有团块纷纷散去，并注意最终体积。 </li><li>再涡流各浆料并立即吸取150微升到每个6孔96孔深孔块（ 图1）上。重要的是要彻底涡流和频繁，以保持土壤的颗粒在悬浮液中。离开每块至少有两个井空作为底物的控制。注:剪辑的移液器吸头与之前的移液剪刀年底的土壤泥浆容易堵塞的提示。准备一台96井区的每种酶进行检测。 </li><li>倒入大约5毫升的醋酸缓冲液入吸管水库和使用8道移液器到150微升的缓冲液加入至升AST各样品2孔（这将样品缓冲液对照组）和2基板对照孔（ 图1） </li><li>倾大约10毫升正常 P NP-底物溶液成吸管水库和使用8道移液器至150微升底物溶液加至前四个孔中每一个样品和两个基板对照孔（ 图1）。注意时间作为反应只要加入底物溶液开始。 </li><li>孵育板在室温（22℃）为0.5-4小时。确切的温育时间将取决于样品中的活性水平和待测定的酶而有所不同。对于大多数土壤和沉积物，磷酸酶和β-葡萄糖苷酶需要0.5-1.5小时的孵育时间，而NAG酶和其他酶需要&gt; 2小时孵育时间。 </li><li>虽然试验是孵化制订明确的96孔微孔板读吸光度。准备一台微孔板的每个深孔块（ 即每封nzyme测定）。移液管加入10μl的1M NaOH和190微升蒸馏水到微孔板的每个孔中。注意:氢氧化钠减慢酶促反应，并且提高，从而提高了反应过程中释放的P NP的颜色的pH值。 </li><li>培养后，离心分离96孔块在2,000-5,000×g离心5分钟以沉淀土壤颗粒。 </li><li>使用多道移液器从每孔撤出100微升，请小心操作以免颗粒，并将其传输到编制明确的96孔板对应得很好。 </li><li>打开酶标仪，并设立任何必要的软件。在410nm处记录吸收值。如果一个特定的孔的吸光度高于酶标仪的线性检测限，淡化了良好1:1的水和重新测量。如果吸光率仍然太高，该测​​定应反复用更短的孵育时间。 </li></ol> 4。样品的干质量的测定<ol><li>移液器各1ml SAMPL的<html> Ë浆到一个预先称重的铝盘中。 </li><li>干在75℃烘箱中干燥48小时后称量。从这个值减去锅的重量，得到土壤或沉积物的干质量的1毫升的浆料。通过0.15的因子相乘，以确定在150微升的酶测定加入到每孔样品的干质量。 </li></ol> 5。每个土壤干质量或沉积物酶活性的计算<ol><li>减去从样品测定吸光度采样控制吸光度计算出每个样品的最终吸光度。如果基板的控制有较高的吸收率（约&gt; 0.060），然后减去者也。 </li><li>计算酶的活性在微摩尔小时-1 g干质量-1从以下公式计算: </li></ol>酶的活性=最终吸光度/（C X孵育时间×样品干重） 胞外酶活性的天然水域的荧光分析对酶活性的荧光分析幔"&gt; 1。底物的制备，标准和缓冲溶液<ol><li>通过在无菌溶解适当的衬底制备200μM的溶液MUB联的底物（ 如 4 - MUB-β-吡喃葡糖苷，4 - MUB-磷酸盐，4 - MUB-N-乙酰基-β-D-氨基葡糖苷）的（高压灭菌）蒸馏水H 2 O的在无菌的15毫升或50ml离心管中。包装在铝箔管，以排除光线并存储在使用之前冰箱。基材应至少1周是稳定的，如果以这种方式存储。 </li><li>通过使100微米4 -甲基伞形酮在无菌蒸馏水H 2 O的原液准备MUB标准冷藏储存在琥珀色或箔纸包裹的瓶子。在使用前立即由1/10稀释为100μM原液到无菌H 2 O的使10μM的用于酶测定的工作溶液。 </li><li>通过溶解8.4克碳酸氢钠&lt;作好准备的100毫米碳酸氢盐缓冲液的原液子&gt; 3到1 LH 2 O和高压灭菌。根据需要使5毫米的酶的测定工作液稀释此原液的1/20到无菌H 2 O </li></ol> 2。在96孔黑色微孔板组织水样<ol><li>组织微板的每个酶按照图2所示的例子。请注意，以允许足够的复制，标准和控制，这个程序可以测定单个酶的活性达九水样在一个96孔微孔板黑色。 </li><li>倒入大约5毫升第一样品放入吸管水库和使用8道移液器以微升pipette200到所有的水井在微孔板（S）的第1列的。丢弃用过的枪头，并根据需要对每个水样，以填补列1-9重复。 </li></ol> 3。设置样品，标准，淬火和控制基板<ol><li>设置控件占样品，标准秒，基板和淬火在相同的黑色微孔板的样品（ 图2）。 </li><li>样品对照样品含有水和碳酸氢盐缓冲液中，并没有在活性计算中使用，但将演示读取整个实验过程中保持一致。淬火控制包括样品的水和荧光标记的标准量的和用于测量荧光的样品水的衍射。基板和标准控件要么基板联或标准的荧光标记，分别与碳酸氢盐缓冲液的组成。 </li><li>倒入大约5毫升5 mM的碳酸氢盐缓冲液到一个干净的移液管水库。吸取50μL缓冲到微孔板1通过9排D和E形成两个复制的每个样品采样控制井。变化的枪头然后行转移200微升碳酸氢盐缓冲液中，以井10到12 A和H。 </li><li>通过调光或关闭李降低环境照明亮灯作为荧光标准对光敏感。 </li><li>倒入大约5毫升10微米4 - 甲基伞形酮到一个干净的移液管水库。吸取50μL进微孔板1通过行H 12，和成井1到9成排G和F，形成每个样品淬火控制和整体水平控制三个重复。无论是将微孔板在黑暗中或覆盖用不透明的盖子，以减少MUB的光降解。 </li><li>打开的荧光计和设置任何必要的软件以准备加入底物之前阅读。注意:某些荧光灯泡可能需要3分钟以上的预热时间。 </li><li>倒入大约5毫升适当MUB联基板（ 如 4-MUB -磷酸）放入干净的移液管水库。使用12通道移液器吸取到50微升到微孔板1通过A排12，进水井1至9中的行B和C形成三个重复测定每个样品和三个基板的控制。立即出现Ÿ继续执行步骤4.1，记录荧光。 </li></ol> 4。记录荧光<ol><li>基板除了微孔板后立即读出初始荧光。阅读荧光后，孵育微孔板在室温（22℃），无论是在黑暗中或覆盖着不透明的盖子，以减少MUB的光降解。 </li><li>以测量水样品中的最大的潜在酶活性所需的温育时间将依赖于酶浓度的样品内。由于这是未知的测定完成之前，该微板将要读出的多个时刻的步骤。通常情况下，在10〜15分钟的间隔读了1个小时的过程中是可以接受的许多酶，虽然与某些酶的活性非常高的样品可以在10分钟之前见顶。 </li><li>继续阅读荧光微孔板在你指定的时间间隔至少1小时。一定要保持微孔板盖或读数之间的暗秒。 </li></ol> 5。每个供水量酶活性的计算<ol><li>在每个时间间隔中的每个样本计算:平均初始样品的荧光（孔D和E），平均最终样品的荧光（井AC），平均标准荧光（孔H10-11），且平均急冷控制荧光（井FG）。 </li><li>对于每个时间间隔，计算在纳摩尔小时酶活-1毫升-1由以下公式计算: </li></ol>酶活性=（平均样品荧光 - 平均初始样品荧光）/（（平均标准的荧光/ 0.5摩尔）×（平均淬火控制荧光/均值标准的荧光）×（0.3毫升）×（时间以小时为单位）） <ol start="3"><li>检查计算每个时间步骤中的活性值。从最高的活动时间步长决定最终的潜在活性。如果活性值继续增加再后来的时间步长，可能需要，如果活性值下降throug豪特的运行过程中，然后用更短的时间的步骤再次运行。最终活性是在衬底消耗小时-1毫升-1的纳摩尔但可以按比例增加，表示为微摩尔小时-1·L-1。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+scale+patterns+in+microbial+extracellular+enzyme+activity+during+leaf+litter+decomposition+in+a+stream+and+its+floodplain.">Fine scale patterns in microbial extracellular enzyme activity during leaf litter decomposition in a stream and its floodplain.</a> Microbial ecology. 58, 591-598 (2009).</li></ol>

作者宣称，他们有没有竞争的财务权益。

确定各种土壤和沉积物中微生物胞外酶的活性可以有效地洞察养分的矿化与有机物处理17的速率。然而，土壤可以在其水分含量而有所不同，所以标准化活动，以土壤干重是很重要的。这就要求（通常两天）超越了简单的测量酶活性的附加干燥步骤。因此，相对于酶活性的水样，接近瞬时的结果提供了在测定中，在土壤和沉积物酶活性的可靠的检测需要几天。对于一些土壤，它甚至可能是...

微生物如细菌和真菌通过生产胞外酶的取得从复杂的有机化合物的营养物质和碳。这些酶通常水解的聚合物为可以被取入细胞小亚基。因此，在生态层面上，这些微生物胞外酶是负责大部分的养分矿化和发生在自然的环境中有机物的分解。酶，例如纤维二糖水解（CBH）和β-葡糖苷酶是纤维素降解和工作重要一致来催化纤维素水解成葡萄糖1,2，它提供了对微生物的吸收和同化一个可利用的碳底物。酶磷酸酶释放的有机磷易溶于无机磷酸基团，基本上矿化磷酸盐和使其可用于大多数生物3使用。其他酶，如N-乙酰氨基（NAG酶），是的importan吨中几丁质的降解，可以使碳和氮供微生物采集4。 一项所述的程序，用于在自然环境中的微生物胞外酶活性的测定是使用人工对硝基苯基（ 对 NP）相连的基板，该最初被开发，以检测土壤磷酸酶活5的方法。此方法依赖于有色终产物， 对硝基苯酚，当人工基板通过适当的酶水解时释放的检测。 对硝基苯酚的随后可以通过测量其吸光度约为400-410 nm的比色定量。这种方法已经被应用到检测其它酶如NAG酶6，并已应用于各种研究着眼于在土壤和沉积物7-9微生物胞外酶活性。 这是originall一种替代方法Ý用于评估细胞外糖苷酶的活性在水生环境10,11是利用4 -甲基伞形酮（MUB）相连的底物。释放（4 - 甲基伞形酮）的最终产物是高荧光的，并且可以使用与周围四百六十分之三百六纳米的激发/发射设置一个荧光计来检测。各种MUB联的人工基质的情况下，允许至少为许多酶（ 如 β-葡糖苷酶，纤维二糖水解，NAG酶，磷酸酶）的活性的荧光测定作为可使用p NP-衬底比色步骤进行测定。其它微生物的胞外酶，如蛋白降解的亮氨酸氨肽酶，可以使用7 - 氨基-4 - 甲基香豆素（COU）键合底物荧光计进行测定。既MUB-和COU联基板已被用来确定各种陆地和水中的样品12,13中酶的活性。 虽然以前的研究单位描述离子束增强沉积荧光或比色微孔板方法来确定胞外酶的活性14，有必要建立一个清晰的介绍了如何进行这样的试验。在这里，我们展示了进行高通量微孔板技术的使用比色p NP联基板的做法土壤和沉积物，并在使用荧光MUB联基板技术自然水域胞外酶的活性分析程序。我们专注于β-葡糖苷酶，NAG酶和磷酸酶的活性的测量，因为这些酶可以分别连接到碳，氮，磷和骑自行车。然而，此处描述的步骤可以应用到使用不同的人工基质的其他胞外酶的测定。

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS AND MATERIALS&lt;/strong&gt;</td></tr><tr><td>Glacial acetic acid</td><td></td><td></td><td>Various suppliers</td></tr><tr><td>Sodium acetate</td><td></td><td></td><td>Various suppliers</td></tr><tr><td>Sodium hydroxide</td><td></td><td></td><td>Various suppliers</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Nitrophenol</td><td>Fisher</td><td>BP612-1</td><td>Alternates available</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Nitrophenyl (&lt;em&gt;p&lt;/em&gt;NP)-phosphate</td><td>Sigma</td><td>N3234</td><td>&lt;em&gt;p&lt;/em&gt;NP-substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;NP-&amp;#946;-glucopyranoside</td><td>Sigma</td><td>N7006</td><td>&lt;em&gt;p&lt;/em&gt;NP-substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;NP-&amp;#946;-&lt;em&gt;N&lt;/em&gt;-acetylglucosaminide</td><td>Sigma</td><td>N9376</td><td>&lt;em&gt;p&lt;/em&gt;NP-substrate</td></tr><tr><td>Clear 96-well microplates</td><td>Fisher</td><td>12-563-301</td><td>Alternates available</td></tr><tr><td>96-well deep well blocks</td><td>Costar</td><td>3958</td><td>Alternates available</td></tr><tr><td>Aluminum weigh pans</td><td></td><td></td><td>Various suppliers</td></tr><tr><td>Sterile 15 ml centrifuge tubes</td><td></td><td></td><td>Various suppliers</td></tr><tr><td>Sterile 50 ml centrifuge tubes</td><td></td><td></td><td>Various suppliers</td></tr><tr><td>4-Methylumbelliferone</td><td>Sigma</td><td>M1381</td><td></td></tr><tr><td>4-Methylumbelliferyl (MUB)-phosphate</td><td>Sigma</td><td>M8883</td><td>MUB-substrate</td></tr><tr><td>4-MUB-glucopyranoside</td><td>Sigma</td><td>M3633</td><td>MUB-substrate</td></tr><tr><td>4-MUB-&lt;em&gt;N&lt;/em&gt;-acetylglucosaminide</td><td>Sigma</td><td>M2133</td><td>MUB-substrate</td></tr><tr><td>Sodium bicarbonate</td><td></td><td></td><td>Various suppliers</td></tr><tr><td>Black 96-well microplate</td><td>Costar</td><td>3792</td><td></td></tr><tr><td>Pipette reservoir</td><td></td><td></td><td>Various suppliers</td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Centrifuge rotor</td><td>Eppendorf</td><td>A-4-81</td><td>For microplates/deep-well blocks</td></tr><tr><td>Microplate reader</td><td>BioTek</td><td>Synergy HT</td><td>Alternates available</td></tr><tr><td>Microplate fluorometer</td><td>BioTek</td><td>FLx 800</td><td>Alternates available</td></tr><tr><td>8-channel pipettor</td><td></td><td></td><td>Various suppliers</td></tr></tbody></table>

determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays

大部分的养分循环和碳处理的自然环境是通过微生物释放胞外酶的活性。因此，这些胞外酶的活性的测定可以给见解生态系统水平的过程，如有机物分解或氮和磷的矿化速率。环境样品中的胞外酶活性的测定通常涉及暴露样品到人工比色或荧光底物和跟踪底物水解的速率。在这里，我们描述了基于微板的方法，这些方法允许对大量样品的一个短的时间内进行分析。样品被允许与人工基质内的96孔微孔板或深孔微孔板块进行反应，并且酶的活性，随后通过吸收所产生的终产物的荧光或使用典型的微孔板里德确定r或荧光。这种高通量的程序不仅有利于空间上不同的网站或生态系统之间的比较，而且还通过减少每个样品所需的试剂整体体积大大减少这种试验的费用。

土壤和水体沉积物通常有胞外酶的活性明显的水平附着的微生物群落（生物膜），颗粒的表面上生长的结果。 图3显示了本次活动依赖于从第三表层沉积物得到颗粒的大小如何变化在密西西比州北部，美国订单源源不断。先前的研究已经表明，在此流泥沙颗粒的细菌群落可以分为基于他们的社会结构的分子分析三个不同的群体:那些在0.063毫米颗粒，那些在0.125，0.25，和0.5毫米的颗粒，...

Watch this Scientific Journal Video about Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays at JoVE.com

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

基于微板程序描述了用于胞外酶活性的比色或荧光分析。这些程序允许在大量环境样品等活动的管理的时间范围内的快速检测。

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

determination-microbial-extracellular-enzyme-activity-waters-soils

Research

JoVE Journal

Environment

38.8K 次观看.  The University of Mississippi. 基于微板程序描述了用于胞外酶活性的比色或荧光分析。这些程序允许在大量环境样品等活动的管理的时间范围内的快速检测。 

视频: Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

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 Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer&#39;s particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays,&#160;but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.

To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.

潜在土壤胞外酶活性的高通量荧光测量

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be ...

环境科学

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

免疫与感染

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

一个Biomining从宏基因组库的纤维素酶的活性高通量筛选

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

Source: Pokhrel, A., et al. A <a href="https://jove.unicartagenaproxy.elogim.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018).
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.

基于薄层色谱的酶活性测定:一种使用薄层色谱法测定体外焦磷酸激酶活性的技术

Source: Pokhrel, A., et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018).
This ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates

Source: Sch&#252;ckel, J., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/54286">High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits.</a> J. Vis. Exp. (2016).
In this video, we demonstrate a high throughput assay for carbohydrate-active enzyme screening using insoluble chromogenic polymer hydrogel substrates. The enzyme degrades these substrates into a soluble colored product that can be quantified by measuring the color intensity of the product.

基于显色聚合物水凝胶的酶筛选测定:一种使用合成底物筛选碳水化合物活性酶的高通量方法

Source: Sch&#252;ckel, J., et al. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits. J. ...

Assay Techniques

Biochemical assays

Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes

Source: Sch&#252;ckel, J., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/54286">High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits.</a> J. Vis. Exp. (2016).
This video demonstrates an assay to screen enzymes effective in degrading complex polysaccharides in plant biomass using insoluble chromogenic biomass or ICB. The enzymes hydrolyze ICB to release soluble dye-linked oligosaccharides, and the enzyme activity is assessed by colorimetric analysis of the reaction product.

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

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