Catechol Oxidase in a Banana, Lab Report Example

Research Question

The purpose of the lab was to develop an understanding the effect of pH on enzyme activity.  The research question for the lab is ‘How does the level of pH affect the activity of enzymes?”

Background Information

Catechol oxidase is an enzyme that can be extracted from the banana. Catechol is oxidized through a chemical reaction by catechol oxidase in the presence of oxygen.  The current experiment investigates catechol oxidase activity.  During the reaction, catechol is converted to benzoquinone, which is a pigment.  The hydrogens in the reaction combine with oxygen and form water.  The product is responsible for the dark color of fruits.  In a banana, catechol oxidase acts upon the colorless catechol and converts it to a brown color melanin.  This is the brown color observed as a banana is cut and exposed to air.  The process the enzyme undergoes in order to turn the banana brown occurs through a specific process. (Science & Plants for Schools, 1; CSULB)  The chemical reaction is illustrated below:

Hypothesis

The enzyme in the reaction, catechol oxidase  is most active and catalyzes the protein more quickly at a pH 7, neutral pH.  Therefore, it is predicted that the enzyme activity will increase at a neutral pH exhibiting a darker brown color when placed in a buffer solution with a pH 7.  In addition, the activity of the enzyme will decrease when placed in a more acidic or basic solution at pH greater than 7 and less than pH 7.

Variables

Independent Variable: the independent variable is the pH buffer solution.

Dependent Variable: Enzyme activity.

Controlled Variable: Temperature

Apparatus

• 2 Bananas
• 2 Cup Water
• Mortar and Pestle
• Butter Muslin
• Googles
• Fiver 200 ml Beakers
• 50 milliliter flask
• Stirring rod
• Distilled water
• Monosodium phosphate
• Sodium hydroxide
• Balance
• pH meter
• Five Test Tubes
• Test Tube Holder
• Medicine Dropper
• Catechol enzyme
• Thermometer (basic Room Temperature thermometer)

Method

Two bananas were mashed using a mortar and pestle in cups of water to amount to twice the volume of the banana. The mashed banana was filtered using a butter muslin and then stored in a refrigerator for four hours.  Buffer solutions were prepared in order to obtain pH solutions at a pH 2, 4, 6, 7, and 8.  In order to create the buffer solutions, 50 milliliters of distilled water was added to a 200 milliliter beaker.  3 grams of monosodium phosphate was measured on a balance and added to the beaker.  The solution was stirred using a stirring rod until completely dissolved. A pH meter was placed into the beaker and the pH was recorded.  The pH was adjusted using sodium hydroxide. If the pH was below 7, sodium hydroxide was added in order to increase the pH.  If the pH was above 7, hydrochloric acid was added using a dropper until the appropriate pH was reached.  This procedure was repeated for all buffer solutions.  All beakers were labeled with the correct pH.

Five test tubes were obtained and labeled for the corresponding pH values to be added to each test tube.  (Test tube 1=ph2, Test tube 2=pH 4, Test Tube 3=pH6, Test Tube 4=pH 7, and Test Tube 5=pH 8)   Buffer solution was added to one-quarter of the test tube for the corresponding labeled test tube.  A dropper was used to add 10 drops of catechol oxidase extract, obtained previously from the banana, into each of the test tubes.  10 drops of catechol was then added to each of the test tubes.  The test tube color was observed every 5 minutes for a total of 20 minutes.  The change of color for each tube was recorded using a scale from 0 to 5, 5 being a dark brown.   The results were recorded in a Table and a graph was created in order to illustrate the difference in enzyme activity.

Data Collection and Procsesing (Dcp)

Collected Data

The raw data was collected and placed into a table.  Data was collected for each test tube every 5 minutes for a total of 20 minutes.  The results are indicated in Table 1.

Table 1.  Results of Enzyme Activity using a Color Scale in each test tube every 5 minutes after the addition of 10 drops Catechol oxidase extract and 10 drops of catechol to each test tube.  (0=no color, 1=slight color change, 2=light brown, 3=medium brown, 4=brown, 5=dark brown)

Data Processing

Table 2.  Average Color Change over 20 minutes for each pH. (0=no color, 1=slight color change, 2=light brown, 3=medium brown, 4=brown, 5=dark brown)

Errors/Uncertainties

Table 3.  Percent Error.  Percent Error was calculated using “5” as the expected result for a reaction.  (Expected Result – Given Result/Expected Result)*100)

Data  Presentation

Conclusion and Evaluation (Ce)

The results of the experiment support the hypothesis that the enzyme activity would increase at a neutral pH compared to reactions with catechol oxidase in acidic or more basic pH solutions.  The results illustrated a darker brown color at pH seven, which is neutral.  This was the darkest color compared to all other test tubes during the 20 minutes observation, as predicted.    As seen in Table 2, the enzyme activity had the highest average at a pH 7; whereas, the lowest enzyme activity was observed at a pH 8.  The results are also exhibited in Figures 1 and 2.   In Figure 1, the most enzyme activity is observed at pH 7 for all time periods observed.  Figure 2 illustrates the averages reported in Table 2.  In addition, the standard deviations are illustrated as well.  The standard deviations are relatively high for each pH.

Evaluation and Improvement Methods

Since the experiment assigned a number value to color and therefore, the expected number was more or less predicted as 5 without previous research to confirm the use of “5” as the expected result, the percent error was high for each of the pH results for enzyme activity.  However, it should be noted that pH 7 illustrated the lowest percent error compared to the other enzyme activity results at different pH levels.

In order to support the hypothesis with a lower percent error in the results, the experiment should be conducted using quantitative data, such as directly measuring enzyme activity through the quantification  in order to test enzyme activity could result in lower percent error to further support the hypothesis.   In addition, the reduction of human error should be more carefully examined, such as the cross-contamination of droppers.  Furthermore, the total amount of solution used for each test tube should be recorded as well.  In fact, future experiments should contain the same amount of solution for each trial and each test tube.

Works Cited

Csulb.  Lab Exercise: Enzymes I Catechol Oxidase.  Retrieved from: http://www.csub.edu/~kszick_miranda/Lab09_Catechol%20Oxidase.pdf

Science & Plants for Schools. (2014).  Teacher/Lecturer Guide.  Unit: Control and Regulation

(H) : Control of Growth and Development, Environmental Influences Title: The inhibition of catechol oxidase by lead. Retrieved from: http://www.saps.org.uk/attachments/article/261/SAPS%20-%20Inhibition%20of%20catechol%20oxidase.pdf.