This study focuses on the degradation of plant-derived organic matter, specifically through the use of carbohydrate-degrading enzymes on complex polysaccharides. The method involves using dye-tagged biomass as a substrate to evaluate enzyme activity.
Biomass is a plant-derived organic matter primarily composed of complex polysaccharides. Carbohydrate-degrading enzymes act on these polysaccharides to produce smaller oligosaccharides.
To evaluate the degradation activity of an enzyme, begin by taking different insoluble chromogenic biomass - ICB substrates present in the wells of a porous reaction plate. These substrates are obtained from dyeing plant-derived materials with a specific color chlorotriazine dye. The resultant biomass has dye-tagged complex polysaccharides serving as a substrate for enzyme activity.
Now, add a buffer solution containing carbohydrate-hydrolyzing enzyme. The buffer maintains the pH for optimum enzyme activity. Place the reaction plate on a shaker, and incubate. Constant agitation allows the enzyme molecules to reach the target polysaccharide in the ICB substrate.
During incubation, the hydrolyzing enzyme cleaves covalent glycosidic bonds within the long-chain polysaccharides. The reaction produces soluble oligosaccharides linked to dye molecules, which remain in the solution.
Upon completion of degradation, place the reaction plate on top of a product plate and centrifuge the assembly. During centrifugation, the colored solution containing reaction products, passes through the filter at the bottom of the plate, and gets collected in the product plate. Using a plate reader, record the absorbance of the supernatant.
A higher absorbance value denotes the generation of a large number of dye-linked oligosaccharides - indicating the efficiency of the enzyme in degrading the ICB substrate.
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.
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