The purpose of this experiment was to determine how the addition of a substrate (ONPG) would affect enzyme activity and how a change in temperature will affect an enzyme’s activity. : We began by taking 7 test tubes with 4 mL of buffer (potassium acetate) and 1 control with 5 mL of buffer. The 7 test tubes with 4 mL of solution had 1 mL of ONPG at various concentrates (seen in table 1). Then, the enzyme beta-galactosidase was added to each test tube at a concentration of 0.2 units/ml. The tubes were timed for exactly 10 minutes .After the time was complete, 1M of sodium carbonate was added to tubes #2-8 to stop the activity. Approximately 1 mL of each solution was added to a cuvette and then placed in a spectrophotometer set at 421 nm. The spectrophotometer measured the light absorbance or optical density of the solution which is depicted in figure 1. As the substrate concentration was …show more content…
Six test tubes were obtained and labeled. Tube #1 was filled with 5ml of buffer while tubes #2-6 were filled with 4mL of buffer. Then, tubes #2-6 had 1 mL of ONPG (5 mM) added. Before adding the enzymes the tubes were preincubated at various temperatures seen in table 2. Once the tubes reached the temperature being tested 1 mL of enzyme (0.2 units/mL) were added to tubes 2-6. After 10 minutes of activity, 1.5mL of sodium carbonate was added to all of the tubes to stop the enzyme activity. Approximately 1 mL of each solution was added to a cuvette and then placed in a spectrophotometer set at 421 nm. The spectrophotometer measured the light absorbance or optical density of the solution which is depicted in figure 2. As the temperature was increased, the enzyme activity had also increased, however, after 90*C the enzyme activity dropped dramatically because it became denatured. The data supports the direct relationship of enzyme activity and temperature as well as the heat causing the enzyme to become denatured after