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 (ꞵ-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 ꞵ pleated sheets held together by hydrogen bonding that make up the secondary structure. The ⍺ helices and ꞵ 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.