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