Lactase Enzymes Lab Report

BIO 5 Lab Report: Lactase Enzymes

Enzymes are biological catalysts or assistants. Enzymes consist of various types of proteins that work to drive the chemical reaction required for a specific action or nutrient. Enzymes can either launch a reaction or speed it up. The chemicals that are transformed with the help of enzymes are called substrates. In the absence of enzymes, these chemicals are called reactants.
Enzymes are thought to have an area with a very particular shape. When a molecule of the right chemical for that enzyme comes along it will fit exactly into the shape. The region of particular shape is called the active site of the enzyme, this is where the reaction is known too take place. The molecule that the enzyme works on
is known as the substrate. Once the reaction has taken place and the products of the reaction leave the active site leaves it ready for another particle of the chemical. The active site of an enzyme has such a particular shape that there is only one type of molecules that will fit it. This is why certain enzymes are extremely specific in their actions.
Lactase is the enzyme in small intestine that digests lactose.
There are a group of people who after childhood do not fabricate proficient lactase, which can impair the body’s capability to digest milk. These few people are known to be lactose intolerant and suffer from symptoms including cramps, gas, and diarrhea.
Lactase is known to be formed by the body. Dairy products have changeable levels of lactose, which affects how much lactase is required for proper digestion. Ice cream, yogurt, and milk contain a significant amount of lactose - even though for difficult reasons yogurt often does not generate symptoms in lactose - intolerant people.
For the 50 million Americans who have been diagnosed with lactose intolerance, many just avoid dairy at all cost in order for them to prevent any painful side effects. Research shows lactose intolerant individuals normally have a higher health risk for osteoporosis, weak bone density, hypertensions, and several cancers such as breast and colon
cancer.
The purpose of this experiment is to examine the specificity of an enzyme (lactase) to a specific substrate (lactose). It is also to observe the actions of the enzyme and how its shape is important to enzyme reactions. In order to test this experiment we will be using two different sugars such as, lactose and sucrose. Results will be identified using glucose test strips, which will verify the occurrence of glucose in the solution. Given the background information provided, we hypothesized 3 theories. In enzyme specificity lactose would have a high specificity and sucrose would have a low specificity. We predicted that if lactose would have an elevated specificity and sucrose would have a low specificity, then lactase will have no effect on the sucrose because it is not its corresponding enzyme. However, the lactose will break down its substrate glucose. We found out that the sugar and the enzyme corresponded to one another or is specific. In environmental factors on lactase, we hypothesized if the lactase or sucrose is exposed to a higher or lower pH it may affect the way it breaks down as well as the shape of the sugar. We then predicted that the lower the pH the more activity will occur because the acidity will speed up the enzyme reaction. Lastly, we hypothesized that if the lactase or sucrose is exposed to a higher or lower temperature it may affect the way it breaks down as well as the shape of the sugar. We then predicted the one with the higher temperature will affect the lactase.
- Enzyme specificity: Procedure 1
Too start off we took four test tubes and labeled them with (A) milk + lactase, (B) milk + water, (C) sucrose + lactase, and (D) sucrose + water with a writing utensil. For each of the following test tubes we used a plastic pipette and added 2 ml of milk and 2 ml of sucrose to the following test tubes. So for tube A and B we added 2 ml of milk, C and D 2 ml of sucrose. We then set a timer for 2 minutes, but did not start it automatically. Immediately after, we added to test tube A and C, using a pipette 1 ml of lactase, in B and D 1 ml of water. After the timer went off we tested for glucose by placing one glucose test strip, using tweezers, in each tube for about 2-3 seconds and removed it from the solution. We observed the color change for a minute then recorded the amount of glucose that the test strip indicates in table 1 of our worksheet. If there was glucose present or not present we would know that it was either “+” or “-”.
Effects of environmental factors on lactase: Procedure 2
We began this test by labeling 6 test tubes with (A) 4° C, (B) 25° C, (C) 100° C, (D) pH 4, (E) pH 7, and (F) pH 10. Using a pipette, we then added 2 ml of milk into each tube. For tubes A through C, we placed each into the corresponding temperature environments and made note of the time. Test tube A was placed in an ice bucket of 4° C. Test tube B we placed it in a tube rack of 25° C. Test tube C is placed in 100° C water bath. We left test tubes A-C in their corresponding environments. We then prepared for the pH experiment by setting our timers for 2 minutes, although we did not start it just yet. We then quickly, using a pipette added 1 ml of pH 4 lactase to test tube D, 1 ml of pH 7 lactase to E, and 1 ml of pH 10 lactase to F. We started our 2 minute timer right after. At the end of the 2 minutes, we tested for glucose by placing one glucose test strip in each test tube and then removing it. We recorded this data in table 2 on our worksheet. Next, we went back to the temperature experiment and set the timer for 10 minutes, but did not start it. Then we note the time which was 3:30 p.m., of how long the milk was incubating in each temperature before the enzyme was added. After we added 1 ml of lactase using a pipette, to tubes A- C while they were in their temperature environments. Finally, we started the 10 minute timer. At the end of the 10 minutes, we tested for glucose by placing one glucose test strip in each tube and them removing it. Lastly, we recorded our data in table 3 of our worksheet. Enzyme specificity:
This experiment produced a variety of results. For the first result, the test for glucose was positive. The glucose result had a level of 200 and was yellowish green. Glucose was also positive in result 2 as you can see in figure 1. In results C and D looking at figure 1 you can see that glucose results were negative.

Figure 1: Enzyme Specificity

In experiment 2 the result of the first test was positive.
We got a glucose result of 800 which is between the colors green and dark green. The next result was positive as well. The glucose result was 200 and was between the levels of light green and green. Our last result for test 2 as you can see on figure 2 was in fact negative, proving that the pH levels can affect the lactase activity.

Figure 2: Effects of pH on lactase activity

In the last experiment, by looking at Figure 3, you can see that the result for glucose was typically low. For our first result we got a positive level of 50, which was halfway between yellow green and light green. Second result had a level of 200 and was also a positive reaction. Our last result was negative. This proves that the temperature can affect lactase activity.

Figure 3: Effects of temperature on lactase activity

For procedure 1, we hypothesized that lactose will have high specificity and sucrose will have a low specificity. We also predicted that the lactase will have no effect on the sucrose because it is not its corresponding enzyme, however the lactose will break down its substrate glucose. While performing this experiment we realized that our hypothesis and prediction was in fact correct. As mentioned before, the glucose level in lactose was high and the sucrose was low. This meant that the sugar and enzyme corresponded to one another or is
specific.
In procedure 2, we hypothesized that if the lactase or sucrose is exposed to a higher or lower pH it may affect the way it breaks down as well as the shape of the sugar. We also predicted that the lower the pH level the more activity will occur because the acidity will speed up the enzyme action. While performing this experiment we realized that once again our theory was right. Results showed that the pH 4 was the most acidic and had the highest effect on lactase activity.
Our last procedure, we hypothesized that if the lactase or sucrose is exposed to a higher or lower temperature it may affect the way it breaks down as well as the shape of the sugar. We also predicted that the one with the larger temperature will affect the lactase. In this case looking at our final result of the glucose test we were incorrect. The temperature that actually had the most affect was the 25°C. We realized that the lactase activity was only affected by a colder temperature or a normal temperature. We came to the conclusion that lactase activity is not affected by temperatures that are at boiling points, such as 100°C.
In conclusion, this lab experiment proved that lactose is in fact affected by both pH and temperature. It also proves that there has to be a specific substrate for lactase in order for the enzyme to produce galactose/glucose.

References:
1. A Description of Lactase and Lactose Reactions. (2011). Retrieved April 8, 2013, from http://www.livestrong.com/article/408258-a-description-of-lactose-and-lactase-reaction/ 2. "Enzymes". (2009). Retrieved April 8, 2013, from

3. Kretchmer, Norman. (1993).
“Lactose Intolerance and Malabsorption in the Cambridge World History of Human and Disease”. Cambridge and New York