We would like to thank Prof. J. Paul Knox (University of Leeds, UK), who provided access to his laboratories for filming, and Susan E. Marcus for excellent technical assistance. JS acknowledges the WallTraC project (European Commission Seventh Framework Program (grant agreement no: 263916) and the project Biomass for 21st century (Innovation foundation Denmark; case no.: 103408). SKK is thanking the SET4Future project (Danish Strategic Research Council), the Bio-Value Strategic founded by the Danish Council for Strategic Research, the Danish Council for Technology and Innovation (Grant Case no.: 0603-00522B), and A Biology-driven Approach for Understanding Enzymatic Degradation of Complex Polysaccharide Systems (Grant Case no.: 107279) for funding. This paper reflects the authors&#8217; views only. The European Union is not liable for any use that may be made of the information contain herein.

1. Chromogenic Assay with CPH Substrates in a 96-well Plate Format
<ol>
	<li>Activation of the assay kit plate
	<ol>
		<li>Activate the 96-well filter (containing blue CPH-xylan) assay kit plate (List of Materials) by adding 200 &#181;l activation solution (obtained with the kit) into each well, followed by 10 min incubation at room temperature without agitation.</li>
		<li>Apply vacuum using a vacuum manifold (with the spacer block inside and any standard, transparent 96-well plate as a collection plate) to remove extant activation solution. It is also possible to use a centrifuge at 2,700 x g for 10 min for this step instead of the vacuum manifold.</li>
		<li>Wash the CPH substrates by adding 100 &#181;l sterile water and apply vacuum (or centrifugal force) to remove the stabilizer. Repeat this step two more times and the plates are now ready to use.</li>
	</ol>
	</li>
	<li>Enzyme reaction 
	NOTE: Always include buffer alone as a negative control and if possible previously characterized enzymes as positive controls. Use a statistically appropriate number of replicates. The CPH substrates are stable between pH 3.0 to 10.0 and the total volume of buffer end enzyme solution in each well should not exceed 180 &#181;l. Plant extract or culture broth can also be used instead of purified enzyme solution.
	<ol>
		<li>Add 150 &#181;l 100 mM sodium acetate buffer, pH 4.5 and 5 &#181;l endo-cellulase solution (to a final concentration of 1 U/ml) to each well of the assay kit plate.</li>
		<li>Put the product plate (a clear-well plate compatible with the microtiter-plate reader) underneath the assay kit plate to collect any potential leakage from the reaction plate during shaking.</li>
		<li>Incubate the assay kit plate at 25 &#176;C for 30 min in a horizontal shaker at 150 rpm. 
		NOTE: Mixing the reaction in the assay kit plate during the incubation is crucial for achieving a consistent and reproducible result. CPH substrates are stable up to 90 &#176;C. The incubation time should be increased up to 24 hr when testing culture broths containing unknown enzyme concentrations with CPH substrates. Note that appropriate incubation times depend on the activity of the enzyme(s), but in general if there is no detectable activity within 24 hr, it is likely that the enzyme will not degrade the tested substrate. An active enzyme is degrading the insoluble chromogenic polysaccharides of the CPH substrate into soluble chromogenic oligosaccharides, which are visible as colored supernatant.</li>
		<li>Place the clean product plate inside the vacuum manifold with the spacer block inside.</li>
		<li>Place the assay kit plate on top and apply vacuum (maximum negative pressure of -60 kPa). It is also possible to use a centrifuge at 2,700 x g for 10 min. 
		NOTE: The filtrate containing the colored oligosaccharides as reaction product is now in the product plate for further analysis8.</li>
	</ol>
	</li>
	<li>Detection and Quantification
	<ol>
		<li>Check that the volume of liquid in each well of the product plate is approximately the same by visual inspection.</li>
		<li>Read the absorbance of the collection plate at 595 nm for blue CPH-xylan using a plate reader.</li>
		<li>When doing data analysis, subtract the buffer-only negative control values from the values from the wells where an enzyme was added. Calculate the mean value and the standard error of means (SEM) from the replicate wells8.</li>
	</ol>
	</li>
</ol>
2. Chromogenic Assay with ICB Substrates in a 96-well Format
<ol>
	<li>Enzyme reaction
	<ol>
		<li>Add the 150 &#181;l 100 mM sodium acetate buffer, pH 4.5 and 5 &#181;l 31 U/ml endo-xylanase solution to each well of the assay kit plate containing red ICB-wheat straw (final enzyme concentration in the well: 1 U/ml). 
		NOTE: The assay kit plates (96-well filter plates containing ICB substrates) are manufactured as described in literature (see List of Materials). The ICB substrates are stable in buffers with a pH range between pH 3.0 to 10.0. Always include buffer alone as a negative control, commercial enzymes as a positive control and use a statistically appropriate number of replicas.
		<ol>
			<li>Do not activate the ICB substrates like the CPH substrate plate, but remove the stabilizer by washing three times with 100 &#181;l water followed by vacuum filtration or centrifugation.</li>
		</ol>
		</li>
		<li>Put the product plate underneath the substrate plate to collect any potential leakage from the substrate plate during shaking.</li>
		<li>Incubate the reaction at 25 &#176;C shaking at 150 rpm for 2 hr. 
		NOTE: An active enzyme is degrading the insoluble chromogenic polysaccharides in the ICB substrate into soluble oligosaccharides, which are visible as colored supernatant. ICB substrates are stable up to 90 &#176;C. The incubation time should be increased up to 24 hr if non-purified enzymes such as culture broths are used.</li>
		<li>Place the product plate inside the vacuum manifold with the spacer block inside.</li>
		<li>Place the assay kit plate on top and apply vacuum (maximum negative pressure of -60 kPa) or use a centrifuge to filtrate the product from the assay kit plate into the well of the product plate. 
		NOTE: The filtrate containing the colored oligosaccharides as reaction product is now in the collection plate for further analysis8.</li>
	</ol>
	</li>
	<li>Detection and quantification
	<ol>
		<li>Check that the volume of liquid in each well of the collection plate is approximately the same by visual inspection.</li>
		<li>Read the absorbance of the collection plate at 517 nm for red ICB-wheat straw using a plate reader.</li>
		<li>When doing data analysis &#8212; subtract the buffer &#8212; only negative control values from the values from the wells where an enzyme was added. Calculate the mean value and the standard error of means (SEM) from the replicate wells8. 
		NOTE: In case of screening unknown enzymes, we suggest making a dilution series in order to obtain more detailed data about the dynamic range of enzyme&#39;s activity.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lombard, V., Ramulu, H. G., Drula, E., Coutinho, P. M., Henrissat, 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+carbohydrate-active+enzymes+database+(CAZy)+in+2013.">The carbohydrate-active enzymes database (CAZy) in 2013.</a> Nucleic Acids Res. 42 (Database issue), D490-D495 (2014).</li><li>Cantarel, B. L., 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=The+Carbohydrate-Active+EnZymes+database+(CAZy):+an+expert+resource+for+Glycogenomics.">The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics.</a> Nucleic Acids Res. 37, D233-D238 (2009).</li><li>Agblevor, F. A., Murden, A., Hames, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+of+analysis+of+biomass+sugars+using+high-performance+liquid+chromatography.">Improved method of analysis of biomass sugars using high-performance liquid chromatography.</a> Biotechnol. Lett. 26 (15), 1207-1211 (2004).</li><li>Black, G. E., Fox, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+analysis+of+sugar+monomers+from+complex+matrices+using+chromatography+in+conjunction+with+mass+spectrometry+or+stand-alone+tandem+mass+spectrometry.">Recent progress in the analysis of sugar monomers from complex matrices using chromatography in conjunction with mass spectrometry or stand-alone tandem mass spectrometry.</a> J. Chromatogr. A. 720 (1-2), 51-60 (1996).</li><li>Miller, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Dinitrosalicylic+Acid+Reagent+for+Determination+of+Reducing+Sugar.">Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar.</a> Anal. Chem. 31, 426-428 (1959).</li><li>Somogyi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notes+on+Sugar+Determination.">Notes on Sugar Determination.</a> J. Biol. Chem. 195 (1), 19-23 (1952).</li><li>Zantinge, J. L., Huang, H. C., Cheng, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplate+diffusion+assay+for+screening+of+beta-glucanase-producing+microorganisms.">Microplate diffusion assay for screening of beta-glucanase-producing microorganisms.</a> Biotechniques. 33 (4), 798 (2002).</li><li>Kra&#269;un, S. K., 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=A+new+generation+of+versatile+chromogenic+substrates+for+high-throughput+analysis+of+biomass-degrading+enzymes.">A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes.</a> Biotechnol Biofuels. 8, (2015).</li><li>Leemhuis, H., Kragh, K. M., Dijkstra, B. W., Dijkhuizen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cyclodextrin+glycosyltransferase+into+a+starch+hydrolase+with+a+high+exo-specificity.">Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity.</a> J. Biotechnol. 103 (3), 203-212 (2003).</li><li>Nyyssonen, M., 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=Coupled+high-throughput+functional+screening+and+next+generation+sequencing+for+identification+of+plant+polymer+decomposing+enzymes+in+metagenomic+libraries.">Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries.</a> Front Microbiol. 4, 282 (2013).</li><li>Sweeney, M. D., Xu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomass+Converting+Enzymes+as+Industrial+Biocatalysts+for+Fuels+and+Chemicals:+Recent+Developments.">Biomass Converting Enzymes as Industrial Biocatalysts for Fuels and Chemicals: Recent Developments.</a> Catalysts. 2 (2), 244-263 (2012).</li><li>Biely, P., 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=Action+of+xylan+deacetylating+enzymes+on+monoacetyl+derivatives+of+4-nitrophenyl+glycosides+of+beta-D-xylopyranose+and+alpha-L-arabinofuranose.">Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glycosides of beta-D-xylopyranose and alpha-L-arabinofuranose.</a> J. Biotechnol. 151 (1), 137-142 (2011).</li><li>Mackenzie, A. K., 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=A+polysaccharide+utilization+locus+from+an+uncultured+bacteroidetes+phylotype+suggests+ecological+adaptation+and+substrate+versatility.">A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.</a> Appl Environ Microbiol. 81 (1), 187-195 (2015).</li></ol>

There are two patent applications filed involving the 96-well plate CPH substrate assay and the 96-well ICB substrate assay (WO2015036000 and Denmark PA 2015 70311).

We are using a new generation of multi-colored CPH and ICB substrates that are based on chlorotriazine dyes (full list of substrates in Table 1) arranged in a custom designed commercial assay kit. Enzyme digestion of the substrates yields small, soluble, dyed products which are detectable in the assay solution and can be quantified using a plate reader9. This assay is designed for evaluation of endo-acting enzymes and the sensitivity of the assay is similar the one using azurine cross-linked (AZCL) substrates10, while other methods are available for exo-acting enzymes11,12. The limitation of this assay kit lies in the detection of endo-enzyme activity, as CPH as well as the ICB substrates are not degradable by exo-enzymes most likely due to steric hindrance arising from the dye and crosslinker molecules8.
The assay is performed in a 96-well format and individual reactions take place in the wells. The reaction has to be mixed in the plate to receive reproducible data. The resulting supernatants are filtered into a product plate where the absorbance of each well can be quantified using absorbance spectrometry. The basic principles and the layout of the assay are shown in Figure 1. The assay consists of an assay plate (a 96-well filter plate) with the substrates, and after incubation with enzymes, the supernatant is filtered through into a clear well plate and the absorbances are read providing a semi-quantitative measurement of enzyme specificity and activity. It has been shown, that this screening assay using CPH substrates can be also used in an agar plate format, where the soluble reaction products create a colored halo after overnight incubation.8
The assay kits can be used to screen purified enzymes and their potential side-activities as demonstrated in Figure 2. Side-activities can arise from a single enzyme and its promiscuous specificity but also from a fact that the sample analyzed is a mixture of different enzymes and their synergic effect needs to be studied. Additionally, as it has been shown in previous studies, that enzyme cocktails, gut microbiota13 and culture broths from fungi8 as well as endogenous plant enzyme and bacteria (unpublished data) can be used as an enzyme source.
ICB substrates address complex mixtures of cell wall components often encountered in industrial processes of biomass breakdown. These substrates are designed to evaluate polysaccharide availability and provide information about how to effectively optimize degradation cocktails for more efficient degradation output. As illustrated in Figure 3 CPH and ICB substrates can be used in enzyme screening side by side &#8212; revealing a wealth of information about enzyme specificity and activity in both the context of the preferred substrate (CPH) and a more natural complex containing other components in addition to the preferred substrate more closely mimicking a macromolecular assembly found in nature (ICB). The use of multiple colors allows for simultaneous detection of different enzyme activities against several substrates which increases the high-throughput and multiplexity of the assay. Spectra of different dyes can be resolved by simple linear regression and in most cases multi-substrate activity can be observed by visual inspection alone. A simulated example of such an experiment and its results is depicted in Figure 4.
This assay toolbox and the versatility of its application are very well suited for first-level screening of enzymes and culture broths with unknown activities. The most important aspects of this assay are its high-throughput nature, customizability, ease of use and flexibility. With that in mind, we believe that this novel set of tools will greatly improve and speed up enzyme screening processes in industrial as well as academic applications.

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.

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:427px;">assay kit plates</td>
			<td style="width:145px;">Glycospot</td>
			<td style="width:155px;"></td>
			<td style="width:288px;">customized assay kit plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">activation solution</td>
			<td>Glycospot</td>
			<td></td>
			<td>for activating CPH substrates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">350 ml receiver plate spacer block for vacuum manifold</td>
			<td>Pall Corporation</td>
			<td>5015</td>
			<td>spacer block</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well MultiScreen HV filter plate, 0.45 &#181;m, clear, non-sterile</td>
			<td>Millipore</td>
			<td>MSHVN4510</td>
			<td>assay plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-Well Microplates, Polypropylene</td>
			<td>Greiner Bio-One</td>
			<td>651201</td>
			<td>collection plate after washing the substrates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nunc&#8482; MicroWell&#8482; 96-Well Microplates</td>
			<td>Thermo Scientific</td>
			<td>269620</td>
			<td>product plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diaphragm pump MZ 2 NT</td>
			<td>Vacuubrand</td>
			<td>732000</td>
			<td>vacuum pump used with the vacuum manifold</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Infors HT Ecotron</td>
			<td>Infors HT</td>
			<td>4950132 (Buch &#38; Holm)</td>
			<td>horizontal shaker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SpectraMax M5</td>
			<td>Molecular Devices</td>
			<td>10067-750 (VWR)</td>
			<td>96-well plate absorbance reader</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacuum manifold</td>
			<td>Pall Corporation</td>
			<td>5017</td>
			<td>vacuum manifold</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">endo-cellulase (EGII) (Trichoderma longibrachiatum)</td>
			<td>Megazyme</td>
			<td>E-CELTR</td>
			<td>cellulase [cel]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">endo-&#946;-1,4-mannanase (Cellvibrio japonicus)&#160;</td>
			<td>Megazyme</td>
			<td>E-BMACJ</td>
			<td>mannanase [man]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">endo-&#946;-1,3-glucanase (Trichoderma spp.)</td>
			<td>Megazyme</td>
			<td>E-LAMSE</td>
			<td>&#946;-glucanase [glu]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">endo-b-1,4-D-galactanase (Aspergillus niger)&#160;</td>
			<td>Megazyme</td>
			<td>E-EGALN</td>
			<td>galactanase [gal]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">endo-&#946;-1,4-xylanase M4 (Aspergillus niger)&#160;</td>
			<td>Megazyme</td>
			<td>E-XYAN4</td>
			<td>xylanase [xyl]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">endo-xyloglucanase (GH5) (Paenibacillus sp.)&#160;</td>
			<td>Megazyme</td>
			<td>E-XEGP</td>
			<td>xyloglucanase [xg]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#945;-amylase (Bacillus licheniformis)&#160;</td>
			<td>Megazyme</td>
			<td>E-BLAAM</td>
			<td>amylase [amy]</td>
		</tr>
	</tbody>
</table>

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.

The high-throughput and multiplexing capacity of this assay is based on insoluble chromogenic polymer (or protein) hydrogel (CPH) substrates arranged in 96-well filter plates. Enzymes as well as negative controls are added to the assay kit plate (Figure 1A) and the enzymes degrade the corresponding substrate producing a colored supernatant (Figure 1B). After the reaction is finished, the supernatant is transferred into a clear-well product plate and the absorbance can be measured directly using a spectrophotometer suitable for 96-well plates (Figure 1C).
An example of a dose response of CPH-arabinoxylan to xylanase at different concentrations of the enzyme (0.00 - 0.75 U/ml) is shown in Figure 1D where the decreasing enzyme concentration can be observed visually. A more detailed spectrophotometric quantification can be used to plot the absorbance versus enzyme concentration (Figure 1E). The signal intensity corresponds to the enzyme activity. The reproducibility of the assay is shown by the error bars (standard error of mean, SEM, of three replicas). More detailed experiments on the reproducibility of this assay are published elsewhere8.
<img alt="Figure 1" src="/files/ftp_upload/54286/54286fig1.jpg" /> 
Figure 1. Xylanase treatment of CPH-arabinoxylan. A) A scheme of the assay kit plate with the CPH substrate (e.g., CPH-arabinoxylan) loaded into 96-well filter plate wells just before the addition of enzymes 1 and 2 and the buffer-only control (enzyme 1 had endo-xylanase activity); B) Degradation of CPH-arabinoxylan by enzyme 1 produced a colored supernatant; C) Upon vacuum-assisted filtration of the supernatants in to the product plate, the absorbance is measured spectrophotometrically; D) product plate containing the reaction products after treatment of CPH-arabinoxylan in 4 different colors with different concentrations of endo-&#946;-1,4-xylanase in 100 mM sodium acetate buffer, pH 4.5 for 60 min at room temperature; E) Quantification of the reaction products from D using spectrophotometry. <a href="https://www.jove.com/files/ftp_upload/54286/54286fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are different options for using this assay in enzyme screening. One option is to use a 96-well plate containing different polysaccharides for screening, e.g., a small number of (purified) endo-enzymes with unknown activity. In this case the result will show which polysaccharides are degradable by the target enzyme. To show this principle, an endo-cellulase was tested against different CPH substrates at 25 &#176;C. Three different enzyme concentrations (0.5 U/ml, 1.0 U/ml and 5 U/ml) were incubated for 30 min. The result is clearly visible in the product plate (Figure 2A). The product sheet for this endo-cellulase provided by the supplier specifies side-activity for xyloglucan (tamarind), barley &#946;-glucan, glucomannan, birchwood xylan and low side-activity for galactomannan. Consistent with this, activity additional to cellulase was found against CPH-&#946;-glucan (barley), CPH-xyloglucan (tamarind), CPH-xylan (beechwood) and low activity against CPH-galactomannan (Figure 2B). Glucomannan was not tested. The same CPH substrates were digested with commercial available enzymes (three different enzyme concentrations: 0.1 U/ml, 0.5 U/ml and 1.0 U/ml) used as positive controls under the same conditions than the previous experiment. All substrates were degraded by the positive control enzyme and the signal intensity increased corresponding to higher enzyme concentration (Figure 2C).
<img alt="Figure 2" src="/files/ftp_upload/54286/54286fig2.jpg" /> 
Figure 2. Eight different CPH substrates were incubated under agitation at 25 &#176;C for 30 min. A) The product plate of different CPH substrates digested with an endo-cellulase, at different concentrations. B) Quantification of the activity and various side activity of the endo-cellulase. The error bars represent the standard error of mean of three replicas. C) Activity of different commercial enzymes to the corresponding CPH substrate (endo-cellulase and 2-HE-cellulose; E-LAMSE and CPH-pachyman, CPH-curdlan, CPH-&#946;-glucan (barley); E-XYAN4 and CPH-xylan, E-XEGP and CPH-xyloglucan, E-BLAAM and CPH-amylose, E-BMACJ and CPH-galactomannan; all enzymes from Megazyme). The error bars represent the standard error of mean of two replicas. <a href="https://www.jove.com/files/ftp_upload/54286/54286fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The insoluble chromogenic biomass (ICB) substrates are a useful addition to the chromogenic substrate repertoire because they retain in part the natural arrangement of polysaccharides in plant cell walls which are the major constituent of biomass. CPH and ICB substrates are used in our example to analyze the secreted enzymes of Phanerochaete chrysosporium when cultivated in liquid medium. The plate setup of the assay kit plate is shown in Figure 3A, with 19 CPH substrates and 5 ICB substrates (4 wells for each substrate, Figure 3B). P. chrysosporium was cultivated for three days and then the culture supernatant analyzed. Therefore, 125 &#181;l 200 mM buffer was transferred to each well and 25 &#181;l culture supernatant added. Three different pH conditions have been tested using sodium acetate buffer pH 4.0, sodium phosphate buffer pH 6.0 or pH 8.0 (Figure 3C). The plate was incubated shaking (150 rpm) at 25 &#176;C for 2 hr.
The reaction products were transferred to the product plate (Figure 3D) and analyzed. P. chrysosporium produced enzymes for the degradation of various glucans, starch and xylans (Figure 3E). Lower signals could be detected for the hemicelluloses arabinan (sugar beet) and pectic galactan as well as for RGI (soybean). The enzymes produced were more active in acidic conditions (pH 4.0) than in neutral or slightly basic conditions (pH 8.0). Lower activity towards ICB substrates (Figure 3F) demonstrates that when the polysaccharides are in a more natural context, the efficiency of the enzyme is not the same as with a pure polysaccharide and that is why ICB substrates demonstrate a more realistic view on enzyme efficiency, if it was applied to raw or pre-treated plant material.
<img alt="Figure 3" src="/files/ftp_upload/54286/54286fig3.jpg" /> 
Figure 3. Screening of a culture supernatant from a 3-day old liquid culture of Phanerochaete chrysosporium using a multi substrate plate containing 19 CPH and 5 ICB substrates. A) A scheme of the plate setup with 4 wells for each individual substrate (grey background = CPH substrates, orange background = ICB substrates). B) Picture of the assay plate containing the substrate. C) Scheme showing the buffer conditions used in that experiment (200 mM sodium acetate pH 4.0, sodium phosphate pH 6.0 and sodium phosphate pH 8.0). D) picture of the product plate after 2 hr at 25 &#176;C. E) Absorbances were detected at 517 nm and plotted for each individual CPH substrate and F) ICB substrate results for the respective enzymes. <a href="https://www.jove.com/files/ftp_upload/54286/54286fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The chromogenic substrates can be also used to study synergistic effects by using a mixture of different colored CPH substrates in one well and analyzing the reaction supernatant after treatment using single enzymes or enzyme cocktails.
In the following example shown in Figure 4, red CPH-cellulose and yellow CPH-xylan substrates were mixed together at an approximately equal ratio in 96-well filter plate wells. Figure 4A shows the colored reaction products after 1 hr treatment at room temperature with no enzyme (control), cellulose cel (2 U/ml), xylanase xyl (1 U/ml) and a mixture of both enzymes (3 replicates for each approach) in 100 mM sodium acetate buffer pH 4.5. For analysis, the reaction product was quantified spectrophotometrically by scanning the absorbance spectrum from 350 nm to 700 nm (Figure 4B). Often visual inspection alone can give an indication of whether the enzyme is acting on one or multiple substrates, however recorded absorbance spectra stemming from different dyes can also be resolved using simple linear regression8 to give a more accurate indication of the extent of degradation of each substrate from the mixture.
Using CPH substrates as mixtures substantially adds to the throughput of the assay, enabling screening against up to 4 different substrates in one experiment (one well). In the example shown are four different substrates used: blue CPH-&#946;-glucan (barley), yellow CPH-xylan (beechwood), green CPH-amylose and red CPH-pectic galactan (lupin). The layout of the substrate plate is shown in Figure 4C and a picture of the assay plate in Figure 4D. The reaction was performed in 100 mM sodium acetate buffer pH 4.5 for 30 min at 25 &#176;C and 150 rpm. First single enzymes with increasing enzyme concentration were tested with the corresponding CPH substrate (Figure 4E) and the colored supernatant was received as expected in the product plate (Figure 4F, row 1A - 12D). Row E contained the two different CPH substrates yellow CPH-xylan and blue CPH-&#946;-glucan, which were degraded with different ratios of the corresponding enzymes endo-xylanase and endo-glucanase. After the reaction, the result is visible in the product plate: the color of the reaction product was darker green-blue, when more endo-glucanase was present (Figure 4F, 4E-6E) and turned into a lighter yellow-green, when the endo-xylanase concentration increased (Figure 4F, 10-12E). The same is seen in row F, where the two substrates red CPH-pectic galactan and yellow CPH-xylan were degraded with endo-galactanase and endo-xylanase. All four different colored CPH substrate were present (Figure 4F, 1G-12F) and single enzymes degraded the appropriate CPH substrate and by adding additional enzymes a combination of the colored reaction products was received.
<img alt="Figure 4" src="/files/ftp_upload/54286/54286fig4.jpg" /> 
Figure 4. A combination of two different CPH substrates, red CPH-cellulose and yellow CPH-xylan, treated with different enzymes. A) Reaction supernatants after treatment of two substrates with endo-cellulase (cel) or endo-xylanase (xyl) or both enzymes. B) Absorbance spectra of the reaction supernatants. A combination of four different CPH substrates treated with different enzymes. C) Scheme of the substrate plate containing the CPH substrates: blue CPH-&#946;-glucan (barley), yellow CPH-xylan (beechwood), green CPH-amylose and red CPH-pectic galactan (lupin). D) Picture of the assay plate containing the different CPH substrates. E) Scheme of the product plate showing the ratio of the added enzymes (nominal concentrations (NC): glu = 1 U/ml endo-glucanase, xyl = 1 U/ml endo-xylanase, amy = 5 U/ml endo-amylase and gal = 0,5 U/ml endo-galactanase). F) Picture of the product plate after 30 min incubation at 25 &#176;C. <a href="https://www.jove.com/files/ftp_upload/54286/54286fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Substrate</td>
			<td>Source&#160;</td>
		</tr>
		<tr>
			<td>CPH-2-hydroxyethylcellulose</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
			<td>(CPH-2-HE-cellulose)</td>
		</tr>
		<tr>
			<td>CPH-amylopectin</td>
			<td>potato</td>
		</tr>
		<tr>
			<td>CPH-amylose</td>
			<td>potato</td>
		</tr>
		<tr>
			<td>CPH-arabinan</td>
			<td>sugar beet</td>
		</tr>
		<tr>
			<td>CPH-arabinoxylan</td>
			<td>wheat</td>
		</tr>
		<tr>
			<td>CPH-casein</td>
			<td>bovine milk</td>
		</tr>
		<tr>
			<td>CPH-chitosan</td>
			<td>animal origin</td>
		</tr>
		<tr>
			<td>CPH-curdlan</td>
			<td>Alcaligenes faecalis</td>
		</tr>
		<tr>
			<td>CPH-dextran</td>
			<td>Leuconostoc spp.</td>
		</tr>
		<tr>
			<td>CPH-galactomannan</td>
			<td>carob</td>
		</tr>
		<tr>
			<td>CPH-laminarin</td>
			<td>Laminaria digitata</td>
		</tr>
		<tr>
			<td>CPH-lichenan</td>
			<td>Icelandic moss</td>
		</tr>
		<tr>
			<td>CPH-methylcellulose</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>CPH-pachyman</td>
			<td>Poria cocos</td>
		</tr>
		<tr>
			<td>CPH-pectic galactan</td>
			<td>potato</td>
		</tr>
		<tr>
			<td>CPH-pullulan</td>
			<td>Aureobasidium pullulans</td>
		</tr>
		<tr>
			<td>CPH-rhamnogalacturonan I (RG I)</td>
			<td>potato</td>
		</tr>
		<tr>
			<td>CPH-rhamnogalacturonan I (-Gal)*</td>
			<td>potato</td>
		</tr>
		<tr>
			<td>CPH-rhamnogalacturonan</td>
			<td>soy bean</td>
		</tr>
		<tr>
			<td>CPH-xylan</td>
			<td>beechwood</td>
		</tr>
		<tr>
			<td>CPH-xyloglucan</td>
			<td>tamarind</td>
		</tr>
		<tr>
			<td>CPH-&#946;-glucan from barley</td>
			<td>barley</td>
		</tr>
		<tr>
			<td>CPH-&#946;-glucan from oat</td>
			<td>oat</td>
		</tr>
		<tr>
			<td>CPH-&#946;-glucan from yeast</td>
			<td>yeast</td>
		</tr>
		<tr>
			<td>ICB-Arabidopsis</td>
			<td>Rosette leaves from Arabidopsis thaliana Col-0 (adult plant)</td>
		</tr>
		<tr>
			<td>ICB-Arabidopsis seeds</td>
			<td>Arabidopsis thaliana</td>
		</tr>
		<tr>
			<td>ICB-bagasse</td>
			<td>Saccharum officinarum (dried adult plant, stem and leaves)</td>
		</tr>
		<tr>
			<td>ICB-crystalline cellulose (filter paper)</td>
			<td>commercial Whatman 3MM Chr Chromatography paper</td>
		</tr>
		<tr>
			<td>ICB-fenugreek seeds</td>
			<td>Trigonella spp. seeds</td>
		</tr>
		<tr>
			<td>ICB-hemp</td>
			<td>Cannabis spp. (dried adult plant, stem and leaves)</td>
		</tr>
		<tr>
			<td>ICB-lupin seeds</td>
			<td>Lupinus angustifolius seeds</td>
		</tr>
		<tr>
			<td>ICB-pollen P. pratense</td>
			<td>Phleum pratense pollen</td>
		</tr>
		<tr>
			<td>ICB-spruce</td>
			<td>Picea spp. (milled tree trunk)</td>
		</tr>
		<tr>
			<td>ICB-tobacco</td>
			<td>leaves from Nicotiana benthamiana (young plant)</td>
		</tr>
		<tr>
			<td>ICB-wheat straw</td>
			<td>Triticum spp. (dried adult plant, stem and leaves)</td>
		</tr>
		<tr>
			<td>ICB-willow</td>
			<td>Salix spp. (dried adult plant, milled tree trunk)</td>
		</tr>
		<tr>
			<td>ICB-Sorghum</td>
			<td>Sorghum spp. (leaves from adult plant)</td>
		</tr>
		<tr>
			<td colspan="2">*(&#946;-1,4-D-galactan side chains removed with endo-&#946;-1,4-D-galactanase)</td>
		</tr>
	</tbody>
</table>
Table 1. List of available Chromogenic Polymer Hydrogel (CPH) and Insoluble Chromogenic Biomass (ICB) substrates.

Watch this Scientific Journal Video about High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits at JoVE.com

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

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 ...

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Biochemistry

12.9K Views.  University of Copenhagen. 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.

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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...