We will measure the activity of b -galactosidase from E. coli. For E. coli, this enzyme is essential if it is to use the sugar lactose as an energy source. You may have heard about how E. coli regulates the synthesis of this enzyme depending on whether lactose, or a similar compound is in the media. This enzyme is also used as a marker enzyme in research. The sugars lactose, galactose, and glucose are all clear in solution.
Enzymes are proteins with a site for substrate binding and catalysis.
The reason that ONPG works as a substrate to assay b -galactosidase is that ONPG is structurally similar to the natural substrate lactose. ONPG is a colorless compound. Since we measure the rate of ONPG cleavage , and not that of the natural substrate lactose, lactose will behave as a competitive inhibitor of ONPG cleavage. Before you begin the first measurement, follow the steps you used last week to prepare the spectrophotometer to make measurements. Measure the rate of reaction with respect to time Place 3 mls of 0.2 mM ONPG solution in a cuvette.