The overall goal of this chromogenic assay is to screen various enzymes in a high-throughput format against a selection of different chromogenic substrates. This method can help answer key questions in the enzyme discovery field such as finding new enzyme activities for the degradation of bio mass. The main advantage of this technique is that the chromogenic substrates are available in four different colors making this technique high-throughput, extremely reliable and cost effective.
To begin, activate the 96-well filter assay kit plate by adding 200 microliters of activation solution to each well. Incubate at room temperature without agitation for 10 minutes. Use a centrifuge and any standard 96-well plate for collection.
To spin-down the extant activation solution add 100 microliters of sterile water to the chromogenic polymer hydrogel or CPH substrates and apply a vacuum or spin to remove the stabilizer. Repeat the wash two more times. To prepare the enzyme reaction add 150 microliters of 100 mM sodium acetate buffer, pH 4.5 and 5 microliters of endo-cellulase solution with three different concentrations to each well of the assay kit plate.
Place the product plate underneath the assay kit plate to collect any potential leakage from the reaction plate during shaking. Then incubate the assay kit plate at room temperature in a horizontal shaker at 100 rpm for 30 minutes. For receiving reliable data it's necessary to agitate the reaction mixture during the incubation.
Place the assay kit plate with the product plate underneath it in the centrifuge and spin-down at 2, 700 x g for 10 min. To transfer the reaction product into the wells of the product plate. Following the spin, visually inspect the product plate to check if the volume of liquid in each well is approximately the same.
Using a plate reader, read the absorbance of the collection plate at 630 nm for the different green CPH substrates. Analyze and plot the data according to the text protocol. To carry out the chromogenic assay with red insoluble chromogenic biomass or ICB-wheat straw begin by adding 200 microliters of activation solution into each well of the assay plate.
Then, incubate the plate at room temperature without agitation for 10 minutes. Remove the stabilizer by using 100 microliters of sterile water to wash the wells three times. Then, add 150 microliters of 100 mM sodium acetate buffer pH 4.5 and 5 microliters of 10 units per milliliter of different endo-xylanase solution.
Position the product plate underneath the substrate plate to collect any potential leakage from the substrate plate during shaking. Incubate the reaction at room temperature at 100 rpm for two hours. After the incubation place the assay kit plate with the product plate underneath it in the centrifuge and spin-down at 2, 700 x g for 10 min to transfer the reaction product in the wells of the product plate.
Check that the volume of liquid in each well is approximately the same. Then, using a plate reader read the absorbance of the collection plate at 517 nm for red ICB-wheat straw. Carry out data analysis according to the text protocol.
Shown here is an example of a dose response of CPH-arabinoxylan to xylanase at different concentrations of the enzyme where the decreasing enzyme concentration can be observed visually. A more detailed spectrophotometric quantification is used to plot the absorbance against the enzyme concentration. With the signal intensity corresponds to the enzyme activity.
In this example of the assay used for enzyme screening an endo-cellulase was tested at three concentrations against different CPH substrates. As seen here, activity additional to cellulase was exhibited for CPH-glucan, CPH-xylan, CPH-xyloglucan and low activity was seen against CPH-galactomannan. The same CPH substrates were digested with commercially available enzymes used as positive controls under the same conditions.
The results here show that all substrates were degraded at levels that increased with increasing enzyme concentrations. In this experiment, five ICB substrates were included with 19 CPH substrates to analyze the secreted enzymes of Phanerochaete chrysosporium under three different pH conditions. P.chrysosporium enzymes degraded various glucans, starches and xylans.
Lower signals could be detected for the hemicelluloses arabinan and pectic galactan as well as for RGI. The enzymes produced were more active in acidic conditions than in neutral or slightly basic conditions. Once mastered, this technique can be done in less than one hour, if it's performed properly.
While attempting this procedure it's important to remember to mix the reaction during the incubation. After it's development, this technique paves the way for researchers in the field of high-throughput screening to explore novel enzyme activities for biotechnology. After watching this video you should have a good understanding of how to use the screening kit.
