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Effects of Temperature on Beta-Galactosidase’s Efficiency

Introduction
In this experiment the enzyme lactase is studied after exposure to different temperatures to see the effect of temperature on the hydrolysis of lactose. Lactase, a Beta-galactosidase (?-galactosidase), is an enzyme that catalyzes the hydrolysis of lactose. Lactase cleaves the bond of the O-galactosidic bond in the disaccharide lactose into glucose and galactose in the presence of water. Beta-galactosidase is a protein with a tetrameric structure of 4 identical polypeptide chains consisting of 1032 amino acids each1. When an enzyme’s environment is altered by changes in pH or temperature the hydrogen bonds and hydrophobic interactions within the protein can be destroyed, causing the protein to unfold and lose its shape. There are four levels of the structure of a protein : primary, secondary, tertiary, and quaternary structure. The primary structure is the chain of amino acids, this chain then folds into the ? helices and ? pleated sheets held together by hydrogen bonding that make up the secondary structure. The ? helices and ? pleated sheets fold together and are held by hydrophobic interactions as well as disulfide bridges at times. The tertiary structure is the polypeptide which can be a protein on its own or multiple polypeptides can bond to form a quaternary structure2. Denaturing a protein can break the secondary and tertiary structure, but in the process of denaturation the primary structure remains intact3. If the shape of an enzyme is changed the active site will no longer be able to bond to the substrate and act as a catalyst. It is hypothesized that if the lactase enzyme is kept at 37 °C (body temperature) then the hydrolysis of lactose will be more effective than lactase that has been kept at room temperature and if lactase is boiled, then the enzyme will denature and will not hydrolyze lactose. In this experiment the controlled variable is the lactose concentration (200 mg/dL), the independent variable is the temperature of the lactose added to the lactose solutions (37 °C, room temperature and boiled).
Materials and Methods
– Lactose solutions (200 mg/dL)
– Lactase solutions (6 lactaid tabs/ 200mL)
– Room temperature
– 37 °C (body temp)
-Boiled
– Micropipette and tips (100 µL)
– Blood glucose meter and test strips
– Microcentrifuge tubes
– Incubator
Pipette 100µL of lactose into a sampling vessel
Check glucose concentration of lactose with blood glucose meter and record results
Pipette 100µL of lactose into labeled empty microcentrifuge tube
Pipette 100µL of the lactase stored at room temperature into the same tube as the lactose from the previous step
Let sit for 15 minutes, frequently mix
Repeat steps 3-5 for the lactase at 37 °C and the boiled lactase, changing pipette tips after each lactase sample is transferred (return the solution with the 37 °C lactase back into the incubator for the 15 minutes after adding the lactase)
After 15 minutes, transfer the solution from each tube into a separate sampling vessel and test glucose concentration with blood glucose meter and record results
Results
Table 1.- Glucose concentration of lactose solutions after exposure to lactase at different temperatures
Lactase temperature
Glucose concentration
Room temperature
97 mg/dL
37?
102 mg/ dL
Boiled
<20mg/dL*
Lactose solution without lactase
<20 mg/dL*
* Below the level of detection of glucose meter (20 mg/dL)
Discussion
It was hypothesized that if the lactase added to the lactose solution is kept at 37 °C (body temperature), then the hydrolysis of lactose will be more effective than lactase that has been kept at room temperature. And if lactase is boiled then the enzyme will denature and will not hydrolyze lactose in the solution tested. The results of this experiment did support the initial hypothesis in the data shown in Table 1. After collecting the data the percentage of lactose hydrolyzed was calculated using this formula : (Glucose mg/dL) / (200 mg/dL)= x/ 100. The glucose reading from each lactose sample was taken and individually put into this formula, 200 mg/dL is used for the denominator of the formula because the hydrolysis of lactose is a 1mol : 1mol reaction, therefore if there is 200 mg/dL of the reactants (lactose) then there would be 200 mg/dL of the products (glucose) if the lactase enzyme worked at 100% efficiency. Below are the calculations for each solution tested.
Lactase solution kept at 37 °C : (120 mg/dL)/ (200 mg/dL) = x/100 x=60%
Lactase solution kept at room temperature: (97 mg/dL)/ (200 mg/dL) = x/100, x=48.5%
Boiled lactase solution: (<20 mg/dL) / 200 mg/dL = x/100 x= <10%
Lactose solution without lactase: (<20 mg/dL)/ (200 mg/dL) = x/100 x= <10%
Table 2.- Percentage of Lactose Hydrolyzed
Lactase temperature
Percentage of lactose hydrolyzed
Room temperature
48.5%
37?
60%
Boiled
<10%*
Lactose solution without lactase
<10%*
* The data collected for the boiled lactase and lactose solution without lactase was below the level of detection (20 mg/dL). The percentage of these samples was calculated using 20 mg/dL as the glucose concentration with the less- than sign to demonstrate that the actual percentage is known as below 10%.
Figure 1.- Percentage of Lactose Hydrolyzed

* The data collected for the boiled lactase and lactose solution without lactase was below the level of detection (20 mg/dL). The percentage of these samples was calculated using 20 mg/dL as the glucose concentration with the less- than sign to demonstrate that the actual percentage is known as below 10%.
It can be inferred that the lactase that was boiled had 0 mg/dL of glucose as well as the lactose solution without any lactase. When boiled, the lactase denatured and therefore was unable to catalyze the hydrolysis of lactose, this results in the lactose remaining a disaccharide and there not being a presence of glucose in the solution because there was no breakdown of the lactose. The lactose solution without any addition of lactase would not have any glucose when tested because the hydrolysis of lactose is an enzymatic reaction that cannot occur without the presence of lactase and water. To further research on the efficiency of lactase when exposed to different temperatures some changes could be made to perform this experiment again. If this experiment was performed again with multiple solutions of the same variables more quantitative data would be collected. This would allow more calculations to be possible to see the accuracy of the glucose meter used or to see the variation of results with multiple samples of the same lactose temperature added to the lactose solution.
Citations
Carroll, Dusty. “Lesson Plan 4: Getting to Know Lactose.” UPENN, University of Pennsylvania, https://www.sas.upenn.edu/~carrolld/LessPlan4Lactase.pdf.
Reece, Jane B., et al. Campbell Biology / Lisa Urry, Micheal Cain, Steven Wasserman, Peter Minorsky, Jane Reece. 11th ed., Pearson Higher Education, 2016.
Ophardt, C. “Denaturation of Proteins.” Denaturation of Proteins, Elmhurst College, 2003, http://chemistry.elmhurst.edu/vchembook/568denaturation.html.

Relationship Between Molecular Size/ Solute Permeability and the Movement of Water

Data

Abstract
The purpose of our experiment is to explore the relationship between molecular size/ solute permeability and the movement of water into and out of plant cells. To do this we will determine the concentration at which the solute and the cellular concentration of the solute in the cell are equal: the iso-osmotic concentration or the point where 50% of the cells are plasmolyzed. In this experiment we tested the isosmotic point, the point of 50% cell plasmolysis, of Elodea leaf cells at different concentrations of NaCl and KCl to determine which solute has the higher isosmotic point. It is hypothesized that KCl will have a higher isosmotic point then NaCl; therefore, a higher concentration of KCl will be required to plasmolyze half of the cells than concentration of NaCl. One drop of each solute at each concentration being tested was added to a sample of Elodea leaf and let sit. After three minutes, each slide was placed under a light microscope, focused, and analyzed. The number of plasmolyzed cells per 100 cells were counted and recorded (Table 1.0 and 2.0). The percent treatment mean and standard deviation were then calculated for each concentration, and all data was plotted. After much analysis, our hypothesis was seen to be correct, for the isosmotic point of KCl was plotted to be higher than that of NaCl. Our results, therefore, suggest the conclusion that there is naturally more KCl present in Elodea Leaf cells than NaCl.
Introduction
Cells must regulate what molecules enter and exit across the cell membrane. The cell membrane is selectively permeable. Water and some small molecules (depending on their properties) are able to diffuse passively across the membrane. Other molecules require transport through channels or pumps, often requiring an input of energy. The cell membrane is composed of a phospholipid bilayer which provides structure to organelles and cells. Water is critical to life on planet Earth. A cell is composed of 70% water. Water can move in and out of cells through passive diffusion in a process called osmosis. Osmosis is passive movement of water from a region of high solute concentration to lower solute concentration. When placed in a hypertonic environment, water flows out of the cell and the cell shrinks. When placed in a hypotonic environment, water flows into the cell and the cell swells. When using the term hyper- or hypotonic, the reference frame is the cell itself. If a cell is in a hypertonic environment, the solute concentration outside of the cell is higher than the solute concentration within the cell. If the cell is in a hypotonic environment, the solute concentration inside the cell is higher than outside. In an isotonic environment, the solute concentration within the cell and outside the cell is equal. This is the iso-osmotic point. Plant cells are surrounded by a rigid cell wall. When placed in a hypertonic environment, water flows out of the plant cell and the plasma membrane shrinks and pulls away from the cell wall. This phenomenon is referred to as plasmolysis.
Materials and Methods
Materials:
Elodea Leaf
Micro Forceps
Glass slides
Light Microscope
Cover Slips
Kimwipes
Droppers
Pipettor (P20, P200, P1000)
Pipettor Tips
Cell Counters
1 M KCl
1 M NaCl
Eppendorf Tubes (1.5 mL)
Distilled Water
Gloves

Methods:
1 mL dilution- calculate the volume needed of the 2 M concentration of KCl to make desired concentration of KCl (C1V1=C2V2) Determine the amount of water that needs to be added

Experiment the plasmolysis of the Elodea cells with water. This is the negative control group; positive control group- 2M concentration of KCl
Dilutions of 0.2, 0.5, 1.0, and 1.5 M KCl Use a P1000 pipettor to pour the necessary amount of water previously calculated and add in the 2M concentration of KCl respectively

Obtain three samples of Elodea leaf and separate them onto three individual glass slides
Put one drop of one of the dilutions onto each sample of Elodea leaf and cover it with a cover slip Make sure this is done quickly so the Elodea leaf does not dry out!

After three minutes, observe the plasmolysis of the cells of each sample under a microscope and take a picture Three replicates for each treatment

Repeat this process with the remaining four concentrations of KCl
Collecting data – roughly count the number of plasmolyzed cells under each concentration
Perform the experiment a second time with NaCl Use the new data to compare the isosmotic point of both solutes

Results
The graph represents the mean (average) percentage of plasmolyzed cells at varying concentrations of NaCl and KCl. Three replicates were used for each average point plotted (n=3). The error bars represent percent ±STDEV (Standard Deviation). The isosmotic point occurs at 50% cell plasmolysis which is at a concentration of 0.6 M for NaCl and 0.75 M for KCl. Results are statistically significant with a p value less than 0.05.
Conclusion
The results obtained show that the isosmotic point of KCl is higher than that of NaCl. This can be seen by the graph since 50% cell plasmolysis occurs at a concentration of 0.6 M for NaCl and 0.75 M for KCl. A group of Elodea leaf cells require a higher concentration of KCl to become 50% plasmolyzed because there is more KCl already inside the cells than there is NaCl. Therefore, the isosmotic point of Elodea leaf cells is different for different solutes because it depends on how much of that solute is already in the cell. This experiment was designed to calculate the Iso-osmotic point in various solutions and explain the direction of water flow into and out of a plant cell under various conditions. Our data highlights the selective permeable membrane of plant cells and the homeostatic regulation of how much water is allowed to enter and/ or exit the cell based on environmental conditions.
References
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, James D. (1994) Molecular Biology of the Cell (3rd edition). P 90, 478-485.
Bennethum, Todd M. (1992) Membrane Permeability: A Quantitative Approach. Page 212 in Tested Studies for Laboratory Teaching, Volume 14 (Cory A. Goldman, Editor). Proceedings of the 14th Conference of the Association for Biology Laboratory Education (ABLE), 240 pages.

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