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

To carry out the chromogenic assay with red Insoluble Chromogenic Biomass, or ICB-wheat straw, begin by adding 200 microliters of activation solution to 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-millimolar 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, and incubate the reaction at room temperature and 100 RPM for 2 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 minutes 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 nanometers for red ICB-wheat straw.