Liquid Lactase Enzymes Behavior: Lab II and III
Bio182
April Binder
Liquid Lactase Enzymes Behavior: Lab II and III
Bio182
April BinderIntroduction
Lactase (β-galactosidase) is a membrane bound enzyme, found in the epithelial cells within the jejunum of the small intestine of mammals that digests the disaccharide lactose (Sharma et al 2007). This enzyme cleaves the lactose into two monosaccharides, glucose and galactose, through a hydrolyzation process (O’Connell and Walsh 2010). The inability to breakdown lactose is mainly found in mature humans and is caused by deficiency of lactase within the small intestine. This phenomena effects nearly three fourths of the human population, where they can experience some fairly uncomfortable symptoms when lactose is ingested (O’Connell and Walsh 2010). One way to help people who suffer from lactose intolerance is to use commercially developed products, such as lactase drops or powder, that help to introduce the enzyme into one’s body to facilitate the breakdown of the lactose within their small intestine making it possible for their bodies to utilize the monosaccharides created. This type of product could be detrimental to helping poor populations across the world gain better access to a predominantly available resource full of nutrition to alleviate some of the starvation and malnourishment issues they currently experience (Sharma et al 2007).
Here, we observe extra strength lactase drops with ortho-Nitrophenyl-β-galactoside (ONPG) as an indicator for enzyme activity, buffers with varying pH balances, and varying temperature conditions, to determine the potential and optimal conditions of the enzyme will be.
Materials and Methods
Chemicals and Instruments
Seeking Health Lactase Drops – Extra Strength, ONPG at varying concentrations, buffers with pH balances ranging from 1 to 9, galactose inhibitors, and a spectrophotometer were all obtained from a lab in Central Washington University, located in Ellensburg WA.
Enzymatic Reactions
On the first day we observed experiments 1 through 3. For the methods of how we prepared experiments 1 through 3, please refer to the instructions located in the lab 2 handout.
A week later we set up experiments 4 and 5 in roughly the same way. In both experiments we set up the spectrophotometer and calibrated it. Then we set up seven clean test tubes. In both experiments we always left tube one “blank”, which meant that it only contained 5mL of the buffer and was set to use as the baseline on the spectrophotometer. The “blank” is treated as identical to the other samples, except that the enzyme has been withheld from it. Experiment 4 contained a buffer with a pH of 5 and experiment 6 contained a pH of 7. Then we added 1mL of ONPG to test tubes 2-7 in both experiments, making sure not to add it to our blank tube and timing when we added it to each test tube. We spaced out the time to add the ONPG to each tube at 15 seconds apart, to keep times consistent. Then we added one drop of liquid lactase to test tubes #3, two drops of lactase to test tubes #4, three drops to test tubes #5, four drops to test tubes #6, and five drops to test tubes #7 (which is the recommended dose for these drops). We mixed each of the test tubes to make sure we got the optimum reaction that was potentially available within each of the conditions within the tubes. We waited 10 minutes for both experiments and then tested the optical density (OD) of each of the tubes, so we could calculate the enzymes activity and find the optimal pH balance along with the optimal amount of drops to use for a catalyzed reaction.
For the last experiment, we set everything up the same as test tube #4 in the fifth experiment. We chose the pH of 7 because it seemed to yield more of an optimal condition between the two pH’s tested. We also chose to use two drops of the liquid lactase, like test tube #4 in the fifth experiment, due to the limitations of the spectrophotometer. We set up four