How We Test for Enzyme Activity
Understanding how enzymes interact with molecules
All enzymes interact with a molecule or compound and produce a change to create a product.
Enzymes work in various ways: They split apart a molecule (like a digestive enzyme breaking down starch), combine two molecules (like DNA polymerase) or add a small group of atoms to a molecule, among many other reactions. Many of these reactions proceed extremely slowly without enzymes present.
When enzymes are present, they dramatically speed up the rate at which the reactions occur.
Preparing the perfect environment for testing
The molecule that the enzyme interacts with is called the substrate. To test enzymes in the lab, we had to provide a substrate, the right chemical environment for the reaction to take place (optimal pH, optimal temperature and the presence of needed minerals, salts or other cofactors for the reaction) as well as a way to measure the product of their action.
For the enzymes we test in foods, there are substrates available that release a yellow color (a nitrophenol or nitroaniline) when they are cleaved by their specific enzyme. The yellow color can then be quantitatively measured by a visible light spectrometer.
To test seven foods for seven assays simultaneously, these assays were done in 96-well plates. Each reaction occurs in a small well with a volume of about 450 µl, or about 0.5 ml (1/10 of a teaspoon). Each enzyme assay takes one 96-well plate.
Our step-by-step procedure to test enzymatic activity