Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates
Transkript
Carbohydrate-degrading enzymes break down the complex polysaccharides into smaller oligosaccharides.
To screen the enzyme degradation activity, begin by taking multiple chromogenic polymer hydrogels, CPH, which are prepared by dyeing polysaccharides with different-colored chlorotriazine dyes. Each polysaccharide is then chemically cross-linked to form hydrogel – a synthetic substrate for the enzymatic degradation.
Now, dispense different CPH substrates into distinct wells of a porous reaction plate. Add an activation solution and incubate it to ensure optimal activation of the substrates.
Place the assay plate over an empty product plate. Centrifuge the plates together at a low speed to facilitate efficient removal of the activation solution through the pores of the reaction plate.
Next, supplement all the wells of the reaction plate with the same type of carbohydrate-degrading enzyme in an appropriate buffer that maintains an optimal pH for enzymatic activity. Seal the plate and incubate it with mild agitation to allow a uniform distribution of enzymes into the polymeric network, allowing them to access the substrate.
During incubation, the enzyme, with appropriate activity against the substrate, degrades the polysaccharides in CPH to produce smaller dye-linked oligosaccharides. These products are soluble, making the solution colored. Spin the plates to collect the colored solution into the product plate.
Spectrophotometrically, determine the product absorbance to measure the enzyme activity.
