Lactase Experiment

Abstract
Salt is a compound formed from the ionic bonding between a negative chlorine atom and a positive sodium atom. When salt is placed inside an aqueous solution, it breaks apart completely. In a solution with lactose and ONPG, they will bind together to catalyze. When all three are placed together, however, salt can denature proteins. In an aqueous solution with lactose and ONPG, they will bind together to catalyze. We hypothesize that in higher salt concentrations, the rate of enzyme reaction will become slower, or decrease. We started by creating our serial dilutions., then made the lactase enzyme. After developing the enzyme, we placed 1 mL of lactase into corresponding cuvette. We let each cuvette to be measured for 300 seconds in

Every concentration (0, 5, 10 and 20%) reached its saturation point. The 10% salt solution displayed the steadiest curve of all concentrations. Of the four concentrations, 10% and 20% salt concentrations show the smoothest curves and lowest absorbance. Of the four different concentrations, the 10% concentration is optimal for enzyme activity compared to the lower concentrations. We reject the null hypothesis, because the higher salt concentrations are, the slower the rate of an enzyme reaction will become. Adding salt to this experiment created a realistic atmosphere of where lactase would function in our bodies. For example, our stomachs consist of acid, salt, etc. To do this experiment again, I would temperature on top of salt concentration to see what the optimal salt concentration would be in an average human body for enzyme activity. Lactase is an enzyme that catalyzes the hydrolysis of lactose into its monosaccharides -galactose and glucose (Russo and Moothart 1986). Lactose can be found in dairy products such as milk cheese, or sour cream. ONPG was the substrate used in our experiment due to it's like structure to lactose (which means it can be catalyzed

We then removed 5 mL from Beaker 1 and placed this into Beaker 2 and added 5mL of our phosphate buffer into that same beaker. We removed 5 mL from Beaker 2 to place into Beaker 3. 5 mL of phosphate was also added into Beaker 3. We again removed 5 mL from Beaker 3 and placed into Beaker 4 with an addition of 5 mL of phosphate buffer. For beaker 4, we removed 5 mL into a ‘waste beaker’ so the volume was equal to Beakers 1-3. This is the end of our serial dilution, leaving us with a 20%, 10%, 5%, and 0% salt concentrations. The 0% salt concentration is our control. We removed 2 mL from each beaker and placed them into corresponding cuvettes. Next we made the lactase enzyme. We crushed a Lactaid pill with a mortar and pestle until it was powder, and mixed that powder with 10 mL phosphate buffer. After that, we filtered the solution into a clean beaker using a paper towel. We then collected 2 mL of that solution into another tube with 10mL of phosphate buffer. After developing the enzyme, we placed 1 mL of lactase into each cuvette. In another beaker, we added 1 mL of lactase in 1 mL of phosphate to create the positive control. Before using the spectrophotometer, we set it to 420 nm and blanked the machine with the cuvette that was filled with only the phosphate buffer. Right before we set the cuvettes into the spectrophotometer, we added 1 mL of ONPG to each