Modification of Enzyme Function in Potato Peel Catalase 

Categories: Enzyme


Catalase is an important enzyme in the cytoplasm of most living cells used to control and lower the build-up of hydrogen peroxide (H2O2), its substrate, by converting it into hydrogen and oxygen molecules. This reaction’s speed, however, is variable to different environmental factors that may affect catalase’s reaction (Mykles et al 2018). This experiment attempts to understand the different ways velocity in a catalase reaction, found in potato peels, can be affected based on different atmospheres. Since catalase enzyme use has been linked to physiological responses when fighting biotic pressures, catalase has a strong importance in the organisms it is present in (Yang and Poovaiah 2002).

Furthermore, its activity variations have been linked to the environmental cues that surround or make its setting, such as type of cell viabilities (Martins and English 2014). It was established in the study that although able to be manipulated, enzymatic reactions are not directed by increasing pH or lack of inhibitor to be faster.

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Hydrogen peroxide is a substrate product that is formed from cells via aerobic respiration. For proper cell function to keep occurring, this compound is broken up by the enzyme catalase in a manner that prevents toxic build-up of H2O2 (Martins and English 2014). This investigation occurs in response to questions Michaelis and Menten might have created when their equation came to life; it is known that enzymatic rate alteration is possible based on environment change, but is it possible to control the rate of reaction and predict correctly how any change can switch the rate of reaction.

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This, being a big part of the enzyme kinetics theorem, explains the relationship an enzyme’s substrate concentration may have to its enzymatic reaction rate (Mykles et al 2018). Further along, the experiment attempted to prove that the catalase enzymatic reaction and the production of oxygen gas and water can be manipulated positively, such as a faster reaction without taking up the reactants in less than a minute, by introducing a higher enzymatic concentration or pH in its environment and by preventing the presence of an inhibitor.

This hypothesis was tested via multiple small experiments over the course of two weeks in which different effectors of enzyme kinetics, such as pH. were manipulated before testing the velocity at which the enzyme, in potato peels, reacted to its substrate. Prior to the performance of this experiment, multiple concentrations of enzymes were tested to obtain a good optimal concentration for its use across the different trials with different environments. This enzyme concentration was also tested with each potato peels source or when obtaining new potato skin juice to confirm that this would be the optimal concentration for different experiments to follow. These experiments then calculated for the most favorable and least favorable environments when pertaining to the effect on reaction rate in the different pressures that affect the reaction for a faster or slower production of O2 and water.

Methods Ideal Enzyme Concentration

The optimal enzyme was first obtained for future parts of the experiments by varying different dilutions of catalase concentration, from grinded up potato peels; by mixing up catalase in 7.2 pH citrate buffer, different dilution percentages (equaling 6 ml each) were obtained while the H2O2 volume was kept constant (1ml) to properly discover the most efficient rate (Mykles et al. 2018). The ideal concentration will run the fastest production of O2 without emptying eudiometer in less than a minute. The final enzyme concentrations tested, which ended up being 25%, 33%, 50% and 75% dilution percentages of enzyme present, were then tested for velocity of reaction to the 1ml of H2O2 via readings of Oxygen production in a time-lapse of 2 minutes (Mykles et al. 2018).. This is done by using a eudiometer setup in which a person reads the meniscus of the bubbles formed by the oxygen released from the reaction, once H2O2 is added, while someone else announces a time interval has occurred (Mykles et al. 2018).

It is important to keep in mind that this method of reading the meniscus give researchers useful data, but it lacks accuracy and precision as the human eye is not fast enough and careful enough to get the best measurement in the few seconds it has. The other members in the experiment time the procedure and notes the rate of the reaction (Mykles et al. 2018). Varying pH Once the optimal enzyme concentration is obtained and established, the rest of the experiments will use this enzyme concentration as the mixture of catalase used. We concluded 33% catalase concentration was our optimal one. Next the rate of reaction is tested at different pH by using different buffers from more acidic to ore basic (5.8 6.6, 7. And 7.8). The procedure is equal to the one followed for the optimal enzyme concentration and only the pH buffers should be different (Mykles et al. 2018).

Varying Substrate Concentration (No Inhibitor)

In this experiment, we diluted and labeled six different hydrogen peroxide concentrations into 3%, 7.5%, 15%, 22.5%, 27%, and 30% H2O2. These were accomplished by a mixture of deionized water and 30% H2O2 to dilute into desired substrate concentrations (Mykles et al. 2018). Then, the same procedure was followed to obtain the info regarding the amount of O2 produced over time at different substrate concentrations. Again, the optimal enzyme concentration found in the first experiment was the one used for this experiment (Mykles et al 2018). This data was used to a Lineweaver-Burk plot (figure 3) to compare with data and Km/Vmax obtained in the next experiment involving change in concentration of substrate in presence of inhibitors. The Km and Vmax of this experiment were also calculated by using the information obtained from the Lineweaver-Burk plot equations in which slope equaled the Km/Vmax and the Vmax equaled 1/y-intercept (Mykles et al. 2018).

Varying Substrate Concentration (With Inhibitor)

Lastly, we tested the rate of reaction of the substrate concentration when presented along an inhibitor, in this case sodium cyanide. The information in this experiment was graphed and calculated as seen in the last experiment and figure 3. The same substrate concentrations were tested as in the last experiment and once again the 33% enzyme concentration was included in the experiment (Mykles et al. 2018). Upon each reaction test, the O2 released per second was also examined again and examined for a correlation with the presence of inhibitor and/or change in substrate concentration.


The experiment done gave a good overview of the way catalase is reactive to hydrogen peroxide and how its rate can be affected. More importantly, environmental changing pressures applied on the enzymatic reaction that catalase and H2O2 cause can affect the velocity of reaction. Upon starting, the optimal enzymatic concentration was obtained by testing the catalase dilutions for efficient production of O2 while reacting for longer than a minute. As seen in figure 1, the 50% and 75% catalase concentrations reacted too fast by finishing all the water in eudiometer before 2 minutes went by to be the ideal concentrations. Along that, figure 1 shows the fast yet controlled way of reaction seen in 33% catalase concentration, in comparison to the slower but so steady 25% concentration, making it the optimal enzymatic concentration.

Therefore, this percentage of catalase was used for the next three experiments regarding the environment changes to the reaction. In the pH testing experimenting, it was observed that a reaction occurring in a pH of 6.8 is much faster and efficient than those results obtained in the other pH presences. Regretfully though, this doesn’t seem to be the actual instance when examining the data that figures 2.A and 2.B portray. In figure 2.A, we are first off able to tell the change in the reaction rate depending on the pH present in the reaction’s environment. The trials indicated in the graph show that the solution with a pH of 6.8 proceeds the fastest while staying over the one-minute mark.

On the other hand, figure 2.B presents the data calculation for the Michaelis-Menten plot and it appears as if the solutions containing pH of 7.2 and up have a more steady and fast rate of reaction. This small discrepancy leads me to believe that the data obtained was not accurate when it comes to the actual amount of buffer added or the time it took for the last two reactions to occur. Sadly, this could be the result of misreading a bubble or performing with the wrong amount of buffer added to the pH mixtures. The change in substrate concentration proved to have a longer more productive effect when lacking inhibitor presence, as seen in figure 3, than when the sodium cyanide inhibitor was present. The graph shows the effect inhibitor and change in H2O2 concentration has on the Km and Vmax of the catalase reaction. From the equations the Lineweaver-Burk plot provide, it was possible to calculate the Vmax and Km obtained when inhibitor sodium cyanide is in solution and when it isn’t.

Composed into Table 1, it can be understood that the presence of inhibitor NaCN causes a smaller Km explaining why there is a higher affinity with lower substrate concentration. The calculations were obtained by multiplying the y-intercept and slope of the equations received in figure 3. Along that, the table shows the Vmax calculation for both reactions. The lack of an inhibitor seems to have a higher value effect to both Km and Vmax. Although not as clear as the other experiments, this also proves that environmental factors do affect the reaction rate and can be manipulated to fit the needs/predictions of the investigating individuals.

To obtain the calculated difference of the standard error possible in the experiment, a t-test was performed with the calculated Km and Vmax values of the reaction with inhibitor and the reaction without inhibitor, as seen in Table 2. The calculated results of everyone doing similar experiments were accounted for to obtain a clear p-value of the likelihood that results are due to chance and error. The resulting p-values (> 0.05) confirmed that both calculations had enough error in them to conclude that there was not enough evidence to back the alternate hypothesis and that the null hypothesis could not be ignored.


The hypothesis of the experiment said that the catalase reaction and formation of O2 could be manipulated to fit a quicker rate of reaction without taking up reactants in less time than a minute, by presenting an increased catalase concentration or pH, and by avoiding the presence of an inhibitor. The results established that the use of increased pH environment and prevention of inhibitor in solution to fit predictions did not support the hypothesis because the t-tests’ demonstrated p-values higher than 0.05 and Km/Vmax values were not expected in the inhibitor-present reaction. This indicated that the null hypothesis might not be rejected and that we don’t have enough evidence to accept our hypothesis.

In contrast, it is safe to say that although results were not as predicted, the fact that any results occurred mean that a change in the environmental factors of an enzyme such as catalase does indeed affect the velocity at which it performs its reactions. A possible reason for why the results for the experiment did not support the hypothesis is the equipment used (eudiometer readings measured by a meniscus of bubble with the naked eye) to collect the measurements in response to the changes made to reactions were not optimal. It is possible that results could be skewed by a misread bubble as some reactions had O2 production so fast that the bubbles were hard to read precisely and on time. If possible, next time this experiment I replicated it is recommended to use a computer-based reader so that the bubble measurements of O2 production will be accurate. The results received in the experiment support the notion that H2O2 is highly regulated in the body of an organism and that catalase existence can be stimulated because there is H2O2 concentration differences in the organism (Martins and English 2014).

Such fact is important and must be regarded when analyzing enzymatic reactions because if not taken under consideration, environmental factors like pH can act as a confounding variable in a study. Overall, though, the experiment showed that enzymatic reactions can be manipulated in different ways to lower or increase important factors like Km.

  • Figures & Tables Figure 1- O2 Evolved Over Time per Enzyme Concentration This figure represents the difference in milliliters of O2 formed from the reaction of catalase to H2O2 over time in seconds at different enzyme concentrations.
  • Figure 2.A- O2 Evolved Over Time per pH Environment This figure represents the milliliters of O2 produced from the reaction over time in seconds at the same enzyme concentration but different pH environment. Figure 2.B- Optimal pH for Catalase (Michaelis-Menten Chart) This figure represents the optimal pH for the enzymatic reaction by showing the better rates of milliliters of O2 produced per second at different pH environments.
  • Figure 3- Substrate Concentration Over Time with and without Inhibitor (Lineweaver-Burk Plot) This figure represents the best rate of reaction for the enzymatic reaction depending on the presence or lack of presence of an inhibitor in the environment.

Literature Cited

  • Martins, D, English, A. (2014) Catalase activity is stimulated by H2O2 in rich culture medium and is required for H2O2 resistance and adaptation in yeast. Redox Biology-Peer Review. Volume 2:308-313
  • Mykles, D, Roberts, E, Pilon, M. (2018) BZ 310 Cell Biology [Laboratory Manual]. Volume 1:ii-112
  • Yang, T, Poovaiah, B. (2002) Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin. PNAS. Volume 99: 4097-4102

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Modification of Enzyme Function in Potato Peel Catalase . (2021, Sep 20). Retrieved from

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