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.
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.
Techniques for mining genomes and metagenomes have developed rapidly in recent years, and so have medium- and high-throughput strategies for cloning and expressing recombinant enzymes. Furthermore, bioinformatic resources and associated depositories, such as (CAZy)1,2 have expanded greatly. However, there are considerable challenges inherent in the exploitation of microbial enzyme diversity for industrial purposes and the empirical determination of enzyme activities has now become a serious bottleneck. For example, it is estimated that, using current methods, we can safely predict the activities of no more than 4% of the proteins within the CAZy database. Although numerous methods are available for monitoring enzyme activities they all have some limitations. Well-established techniques based on chromatography combined with mass spectrometry are available for assessing the oligomeric fragments of glycosyl hydrolase (GH) activities3,4. However, these approaches are labor intensive and generally low-throughput. Methods based on the measurement of reducing sugars such as the dinitrosalicylic acid5 and Nelson-Somogyi6 assays are widely used for assessing GH activities. However, these assays have limited throughput and can be prone to side-reactions. Individual chromogenic polysaccharide substrates, such as azurine cross-linked (AZCL) are widely used for determination of enzyme activities, but purchasing all of the substrates separately and manually distributing the substrate powders within the assay plate can be cumbersome and costly7.
We have developed a new generation of chromogenic polymer hydrogel (CPH) substrates based on chlorotriazine dyes that, when used in conjunction with a 96-well filter plate, form a high-throughput assay system. Additional Insoluble Chromogenic Biomass (ICB) substrates were developed which provide information about substrate availability within complex polymer mixtures, such as those that exist in lignocellulosic biomass. Each substrate can be produced in one of four colors, and different colored substrates can be combined in a single well. In this protocol is shown that this methodology can be applied to a wide variety of polysaccharides and proteins and the potential for screening GHs, lytic polysaccharide monooxygenases (LPMOs) and proteases. Specific protocols are provided for the use of 96 well plates and representative results illustrate the high efficiency of the CPH and ICB substrate kits as tools for enzyme screening.
One significant advantage of the assay kits described, regardless of the substrate, is that the kits are ready to use within 15 minutes, after the activation step. This eliminates the need for time-consuming assembly of the assay from raw substrate materials as it is the case with some other methods7. The CPH and ICB substrates have excellent storage (at least one year at room temperature), pH and temperature stability 8 and require no specialized equipment or training. The CPH or ICB assays are based on 96-well filter plate within which the reaction with the enzyme is conducted. If the enzyme is active with a given substrate, soluble dyed oligomers are generated, producing a colored supernatant which can then be filtered into a regular clear-well 96-well plate using a vacuum manifold or a centrifuge 8.
The substrates are dyed with chlorotriazine dyes which absorb in the visible spectrum (VIS) range and individual colors (red, blue, yellow and green) can be resolved using linear regression if different CPH substrates of different colors are mixed in a single well, and the enzyme acts on more than one substrate. The resulting plate with the supernatants can be measured using a standard microtiter-plate reader capable of measuring absorbance in the VIS range. Mixing different substrates with different colors in one well increases the throughput of the assay system, to a total of 384 experiments in a 96-well plate (4 different substrates of different colors per well).
CPH substrates provide a valuable tool for assessing the specific activity of an enzyme while ICB substrates are used to evaluate the capacity of an enzyme to digest a component within the context of complex substrate mixtures that enzymes usually encounter within biomass. Although ICB substrates do not provide information about individual enzyme specificities, they are nonetheless useful tools for assessing the commercial performance of enzymes, cocktails or broths.
我们使用了新一代多色CPH和ICB基板是基于氯代三嗪染料(在表1基板的完整列表)设置在定制设计的商用测定试剂盒。基板的酶消化产生小的,可溶的,染色的产品,是在测定溶液可检测,并且可以使用读板器9进行量化。该试验被设计用于内切短效酶的评价和测定的灵敏度是类似于使用azurine交联(AZCL)所述一个基片10,而其他方法可用于外短效酶11,12。该测定试剂盒的局限性在于该检测内切 -酶的活性,如CPH以及ICB衬底不能降解的由外 -enzymes最可能是由于从染料和交联剂分子8所产生的空间位阻。
该测定在96-逢执行L格式和个人反应发生在孔中。该反应已在板被混合到接收再生的数据。所得上清液被过滤成产品板,其中每个孔的吸光度可以用吸光度法进行定量。的基本原理和测定的布局示于图1中 。该测定法包括测定板(96孔过滤板)与基板,并用酶温育后,将上清液通过过滤到一个明确的孔板和吸光度读取提供酶的特异性和活性的半定量测量。已经显示,当使用CPH基板此筛选测定所用的琼脂平板格式,其中所述可溶的反应产物创建过夜孵育后的着色卤素可使用。8
该检测试剂盒可用于筛选纯化的酶和它们电位侧活动作为证明图2。侧活动可以从一个单一的酶及其混杂的特异性,但也从一个事实,即所分析的样品是不同酶的混合物和它们的协同作用需要研究产生的。此外,因为它已经在以前的研究已经显示,从真菌8以及内源植物酶和细菌(未发表的数据),该酶鸡尾酒,肠道菌群13和培养液可以使用作为酶源。
ICB基板处理生物质击穿的工业生产过程中经常遇到的细胞壁组分的复杂混合物。这些衬底旨在评估多糖可用性,并提供有关如何有效地优化降解鸡尾酒更有效地降解输出信息。 如图3所示CPH和ICB基板可在酶的筛选并排使用-揭示了大量有关酶的特异性信息在优选的衬底(CPH)和含有除了优选底物更紧密地模仿自然界中(ICB)发现的大分子装配其它部件更自然的复杂的两个上下文D活动。使用多个颜色允许对不同酶活性同时检测针对这增加了测定的高通量和multiplexity几个衬底。不同的染料的光谱可以通过简单的线性回归和在大多数情况下,多基片活性可以通过目测单独观察来解决。这样的实验及其结果的模拟例子在图4中描绘。
此法工具箱及其应用的通用性是非常适合的酶和培养液未知活动第一级筛选。这种测定的最重要的方面是它的高通量的性质,可定制性,易用性和灵活性。考虑到这一点,WË认为,这种新型的工具集,将大大改善和加快工业用酶的筛选流程以及学术应用。
The authors have nothing to disclose.
我们要感谢教授J·保罗·诺克斯(利兹大学,英国),谁拍戏提供给他的实验室访问和苏珊·马库斯优秀的技术援助。 JS承认WallTraC项目(欧盟第七框架计划(赠款协议号:263916),并为21世纪的项目生物质(创新基金丹麦;案号103408)SKK的感谢SET4Future项目(丹麦战略研究理事会)在生物价值的战略由丹麦战略研究理事会,丹麦议会的技术和创新(批准案号0603-00522B)成立,并理解复杂多糖系统(格兰特案例酶降解生物学驱动的方法没有。:107279)资助本文仅反映了作者的观点,欧洲联盟不承担责任对于可以由此处提供的信息包含任何使用。
assay kit plates | Glycospot | customized assay kit plates | |
activation solution | Glycospot | for activating CPH substrates | |
350 ml receiver plate spacer block for vacuum manifold | Pall Corporation | 5015 | spacer block |
96-well MultiScreen HV filter plate, 0.45 µm, clear, non-sterile | Millipore | MSHVN4510 | assay plate |
96-Well Microplates, Polypropylene | Greiner Bio-One | 651201 | collection plate after washing the substrates |
Nunc™ MicroWell™ 96-Well Microplates | Thermo Scientific | 269620 | product plate |
Diaphragm pump MZ 2 NT | Vacuubrand | 732000 | vacuum pump used with the vacuum manifold |
Infors HT Ecotron | Infors HT | 4950132 (Buch & Holm) | horizontal shaker |
SpectraMax M5 | Molecular Devices | 10067-750 (VWR) | 96-well plate absorbance reader |
Vacuum manifold | Pall Corporation | 5017 | vacuum manifold |
endo-cellulase (EGII) (Trichoderma longibrachiatum) | Megazyme | E-CELTR | cellulase [cel] |
endo-β-1,4-mannanase (Cellvibrio japonicus) | Megazyme | E-BMACJ | mannanase [man] |
endo-β-1,3-glucanase (Trichoderma spp.) | Megazyme | E-LAMSE | β-glucanase [glu] |
endo-b-1,4-D-galactanase (Aspergillus niger) | Megazyme | E-EGALN | galactanase [gal] |
endo-β-1,4-xylanase M4 (Aspergillus niger) | Megazyme | E-XYAN4 | xylanase [xyl] |
endo-xyloglucanase (GH5) (Paenibacillus sp.) | Megazyme | E-XEGP | xyloglucanase [xg] |
α-amylase (Bacillus licheniformis) | Megazyme | E-BLAAM | amylase [amy] |