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 Practical 6: Lab Report

SCPBA1-B22

Author: Maryam Hirsi

JUNE 12, 2024

EDUVOS
Faculty of Applied Science

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Abstract
This study investigates the catalase activity under various experimental conditions, including substrate
concentration, pH levels, and temperature variations. The methodology employed rigorous sanitation
procedures and precise experimental tests to ensure accurate results. Results indicate significant
correlations between experimental parameters and catalase activity, shedding light on enzyme behavior
under different conditions. Findings contribute to the understanding of enzymatic mechanisms and
provide insights for further research in enzymology.
Introduction
Enzymes plays a significant role in biochemical processes, catalyzing reactions fundamental to life.
Catalase, an omnipresent enzyme in organisms, facilitates the decomposition of hydrogen peroxide into
water and oxygen. Understanding catalase activity across different conditions is imperative for
elucidating enzyme kinetics and their applications across various domains. This study endeavors to
scrutinize the effects of substrate concentration, pH levels, and temperature variations on catalase activity,
aiming to provide valuable insights into enzymatic behavior.

Methodology
Before the initiation of the experiment, all equipment and stations were thoroughly cleaned and sanitized
to prevent any potential contamination that could have compromised the integrity of the experiment. A
liver was meticulously sectioned into small pieces to facilitate its placement into the test tubes, employing
forceps and scissors for precision.
Experiment 1: Observing Normal catalase activity.
In the experiment, 2ml of 3% hydrogen peroxide was dispensed into a prepared clean test tube, labeled as
"Test Tube 1." Subsequently, the liver piece was introduced into the test tube. The observers proceeded to
monitor the reaction of the hydrogen peroxide with the liver piece, noting the rate at which the reaction
occurred and discerning any variations in the temperature of the test tube. After the observation, all
relevant data were carefully documented.
Experiment 2: Reusing catalase.
A test tube labeled "test tube 2-initial" was prepared by adding a solution of 2ml of 3% hydrogen peroxide
along with a piece of liver tissue. Subsequent steps involved observation and recording of any reactions.
Assuming the reaction had reached completion, the resulting solution was transferred into another test
tube labeled "test tube 2-secondary." Following this, an additional 2ml of hydrogen peroxide was
introduced into the remaining liver within the original test tube labeled "test tube 2-initial." Observations
were made, and data were recorded accordingly.
In the subsequent step, 2ml of hydrogen peroxide was again added to the remaining liver within the
original test tube labeled "test tube 2-initial." The contents were then transferred into another test tube
labeled "test tube secondary part 2." Following this, a further 2 ml of hydrogen peroxide was added to the
contents of the initial test tube. Observations were conducted, and relevant information was recorded.
Experiment 3: Effect of temperature on catalase activity

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In the initial phase of the experiment, a piece of liver was positioned at the base of a test tube, denoted as
"test tube 3," and submerged in a small quantity of distilled water. Subsequently, the test tube was
immersed in a water bath containing boiling water for a duration of 5 minutes. Upon completion, it was
carefully withdrawn from the water bath and allowed to cool naturally, after which the water within the
test tube was removed. Following this, 2ml of hydrogen peroxide was introduced into the test tube with
utmost caution, employing a test tube holder due to the high temperature of the apparatus. Observations
were then conducted to collect any resultant reactions, with the findings documented.

In the subsequent phase of the experiment, two sets of clean test tubes were prepared, each containing an
equal portion of liver tissue. These were respectively labeled as "test tube 3A hot water" and "test tube 3A
ice water." Simultaneously, 1ml of H2O2 was dispensed into separate test tubes, each designated as "test
tube 3B hot water" and "test tube 3B ice water." To distinguish between the sets, one set was subjected to
a hot water bath while the other to an ice water bath, both for a duration of 3 minutes. Following this
treatment, the contents of each test tube were combined with their corresponding counterparts.
Subsequent observations were made and meticulously recorded to catalog any changes or reactions.
Experimental 4: Effect of pH on catalase activity
In the experimental procedure, 2ml of hydrogen peroxide was added to a set of three test tubes, following
which 5 drops of universal indicator were carefully dispensed into each test tube using a dropper. Each
test tube was then meticulously labeled as "test tube 4.1," "test tube 4.2," and "test tube 4.3."
Subsequently, a pH color sheet was provided for future analysis.
In the subsequent steps, specific reagents were added to each test tube to achieve desired pH levels. In test
tube 1, 1 molar of HCl (hydrogen chloride) was incrementally added until the color of the solution
corresponded to a pH of 3, as indicated by the pH color indicator with the assistance of the universal
indicator. In test tube 2, 1 molar of NaOH (sodium hydroxide) was similarly added until the solution
reached a pH of 10. Test tube 3 received a combination of 1 molar of HCl and 1 molar of NaOH until the
solution attained a pH of 7.
Care was taken during the addition of reagents, as an acid can cause skin irritation upon contact. The
contents of each test tube were then cautiously swirled to ensure thorough mixing and attainment of the
desired pH levels. Subsequently, a piece of liver was introduced into each test tube, and the rate of
reaction occurring within the solution was recorded for further analysis.
Experiment 5: effect of substrate concentration on catalase activity
A series of 10ml hydrogen peroxide concentrations, previously prepared by the laboratory technician,
were distributed in five test tubes with the use of a dropper, each labeled according to its concentration
(0%, 2%, 4%, 6%, 8%). Simultaneously, a small piece of liver was placed into each of the five test tubes.
The rate of reaction was then observed over a period of 10 minutes, with the solutions' reactions to the
liver pieces noted at two-minute intervals until the completion of the 10-minute period.

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Results
In this section, a key has been provided to measure the reaction rate, where 0 represents the absence of
bubbling, 1 symbolized the formation of small bubbles, 2 indicates rapid bubbling, and 3 signifies rapid
bubbling or foaming.
Experiment 1: Observing Normal Catalase Activity
Upon placement of the liver piece into the test tube containing hydrogen peroxide, a rapid formation of
bubbles ensued, leading to overflow of the test tube. The rate of bubble formation remained high initially,
gradually declining until cessation. A murky white residue, containing remnants of liver tissue, persisted
in the test tube, accompanied by a warm to slightly hot temperature upon touch. Notably, the substances
within the test tube did not exhibit homogeneity, maintaining distinguishable differentiation.
Experiment 2: Reusing Catalase
Similar to Experiment 1, Test Tube 2-Initial exhibited vigorous bubble formation upon the addition of
hydrogen peroxide to the liver tissue. The temperature of the test tube remained warm to slightly hot, with
a subsequent decline in bubble formation. The resultant liquid in the test tube retained a murky white
appearance, indicative of remaining liver pieces. Upon successive additions of hydrogen peroxide to the
initial test tube, the formation of bubbles decreased significantly, with the temperature of the test tubes
remaining within the same interval throughout. In Figure 1, the initial test tube depicts the state after the
first solution has been poured out and hydrogen peroxide was added. The second test tube illustrates the
condition subsequent to the second addition of hydrogen peroxide in the initial test tube. Figure 1 depicts
the sequential steps involved in the reuse of catalase.

The initial state shows the solution poured into the catalase for the first time, indicating vigorous bubble
formation(Right image). Subsequently, after the first solution is poured out, the catalase is subjected to a
second addition of the solution, leading to a discernible reaction (left image). These visual representations
elucidate the iterative process of catalase reuse and provide insights into the dynamics of enzymatic
reactions over multiple cycles.

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Experiment 3: Effect of temperature on catalase activity

Temperature (°C) Rate of enzyme activity Explanation

0 1 The fluid in the test tube showed
very little bubble formation, and
the bubbles formed slowly.

25 2 .In this test tube, bubbling

formed at a considerably faster
pace and remained active for a
substantial duration.
37 3 In the test tube, a large number
of bubbles were formed and
persisted for an extended period.
100 0 In this test tube, no reaction
occurred
The results of this experiment can be represented in a table format to illustrate the enzyme's rate changes
at different temperature intervals. The key previously mentioned, symbolizing the rate of reaction, will be
utilized in this experiment as well.
Table 1: The temperatures and their rate of reaction and what had been observed in their respective test
tubes.
Experimental 4: Effect of pH on catalase activity In this section of the experiment, utilizing
tabulated results would facilitate a clearer explanation. Table 2: The table explains the different reactions
the pH had to the liver piece

pH Rate of enzyme activity Explanation

3 1 In the test tube, the liver piece
exhibited minimal reaction to
the pH solution. Little to no
bubbles were formed, and the
reaction proceeded at a slow
pace.
7 0 The liver piece did not react to
the pH solution at all. No
bubbles formed.
10 3 In the test tube, the liver piece
reacted vigorously to the pH
solution, resulting in rapid
bubble formation. The tube
became noticeably high in
temperature during the reaction.

Experiment 5: effect of substrate concentration on catalase activity

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In this experiment, employing a table format to illustrate the substrate concentration at various time
intervals would be efficient. Table 3 organizes the substrates concentration levels and the time intervals
the rate of reaction had occurred.

Substrate 0 2 4 6 8 10
concentration minutes minutes minutes minutes minutes minutes
0% 0 0 0 0 0 0
2% 1 1 1 1 1 1
4% 2 1 2 1 1 1
6% 3 3 3 2 1 1
8% 3 3 3 2 2 1

In the test tubes with a concentration of 5%, no bubbles or reaction occurred to the liver piece throughout
the 10 minutes. In the test tube with a 2% concentration, it maintained a constant rate of forming little to
no bubbles and no reaction during this period. For the test tube with a 4% concentration, bubbles formed
instantly at a faster pace initially, but as time progressed, the rate of bubble formation decreased
progressively. Similarly, in the test tubes with concentrations of 6% and 8%, both exhibited rapid
formation of bubbles for a duration of 6 minutes. However, during the last 2 minutes, the rate of bubble
formation decreased.
Discussion
In Experiment 1, the observation of normal catalase activity revealed the formation of hydrogen peroxide
bubbles during the reaction with the liver piece. These bubbles comprised oxygen, a byproduct of the
breakdown of hydrogen peroxide by the enzyme from the catalase (derived from the liver piece) into
water and oxygen upon contact (Lohner, S. and Science Buddies, 2016). This reaction led to the
formation of bubbles. Additionally, an exothermic reaction occurred, resulting in an increase in
temperature as a byproduct (Libretexts, 2021). The chemical reaction between hydrogen peroxide and the
liver piece can be described as a decomposition reaction as shown below:

H2O2 ———–> H2O + O2 catalase

In experiment 2, the continuous reuse of the catalyst with the hydrogen peroxide solution was observed in
the test tube. This observation confirmed the potential for continuous reuse, as catalase decomposed the
solution of hydrogen peroxide (Sapkota, 2022). However, the drawback of this process was identified: the
liver could only be reused a limited number of times before dissolution (Sapkota, 2022). The residue
remaining in the test tube consisted of the water component of hydrogen peroxide and some foam
generated by the released oxygen gas during the reaction (Sapkota, 2022).
Furthermore, this experiment posed certain risks, including exposure to hydrogen peroxide fumigation,
which could result in burns and skin irritation upon contact, and respiratory paralysis if large amounts of
fumigation were inhaled (West, 2023).
In experiment 3 is can be come to conclusion that the environmental temperature change effects the rate
at which the catalyst reaction would occur in the temperature. The most suitable temperature at which the
catalyst could react the most is at the temperature of 37 degree Celsius the temperature at which the
reaction could not occur is at 100 degrees. This is due to the high temperature causes the enzymes in the
liver to denature (Libretexts, 2020). Elevated temperatures cause ionic and hydrogen bond breakage,

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which alters the structure of enzymes (Libretexts, 2020). The disadvantage to this test is the time
consumption each process takes
In Experiment 4, the results indicate a fundamental relationship between pH levels and catalase activity.
Notably, at a pH of 3, the enzyme exhibited minimal activity, resulting in a sluggish reaction pace.
Contrastingly, at a pH of 10, the enzyme displayed rapid activity, characterized by large amounts of
bubble formation. Interestingly, no discernible activity was observed at a neutral pH of 7, indicating a
state of enzyme inhibition. These findings inidcate the pivotal role of pH regulation in effecting enzyme
function, highlighting the necessity for specific pH conditions to optimize enzymatic activity.
The mechanism behind pH's influence on enzyme activity lies in its ability to induce structural changes in
the enzyme, particularly around its active site (AAT Bioquest, 2020). Any deviation from the optimal pH
can perturb the enzyme's conformation, hindering its ability to effectively bind with substrate
molecules(AAT Bioquest, 2020). Consequently, enzymatic activity is diminished, and at extreme pH
levels, the enzyme may undergo denaturation, rendering it functionally inactive (AAT Bioquest, 2020).
This relation between pH and enzyme activity signifies the importance of maintaining an appropriate pH
environment in biological systems to facilitate optimal enzymatic function.
The results of experiment 5 reflect typical enzyme behavior. At lower substrate concentrations, like 2%,
the reaction was steady but limited. As substrate concentration increased, particularly at 4%, the reaction
initially sped up, then slowed down over time. At higher concentrations, such as 6% and 8%, the reaction
was rapid initially but tapered off later. The absence of reaction at 5% concentration suggests it may have
been too low for catalase activity. These findings demonstrate how substrate concentration affects enzyme
activity and emphasize the need for precise experimental control.
Substrate concentration directly impacts enzyme activity. Initially, as substrate concentration increases,
the rate of enzyme-catalyzed reaction also increases simultaneously, as more substrate molecules are
available to bind with enzymes (Libretexts,2021). This relationship continues until all enzyme active sites
are saturated with substrate molecules, at which point further increases in substrate concentration do not
result in a significant increase in reaction rate (Libretexts,2021).. This phenomenon is known as enzyme
saturation. Overall, substrate concentration plays a crucial role in regulating enzyme activity and
ultimately determines the rate of enzymatic reactions.
Enzymes are vital biocatalysts that are essential to many biological and commercial processes. They are
crucial to the pharmaceutical industry's production process because of their efficiency and selectivity, as
enzymes such as lipases and proteases are used in drug synthesis and purification (Smith et al., 2020).
These enzymes facilitate the creation of premium pharmaceutical goods while also streamlining
manufacturing procedures. Enzymes aid in the processes of fermentation, cheese ripening, and starch
hydrolysis during baking, which all improve product quality and shelf life in the food business
(Richardson et al., 2017).
Enzymes are essential for bioremediation, which goes beyond industrial uses to promote environmental
sustainability. Enzymes like dehalogenases and peroxidases are used to break down contaminants in water
and soil, providing a more environmentally friendly option to conventional remediation techniques (Dror
et al., 2021). Furthermore, enzymes play a critical role in the manufacture of biofuels by facilitating the
enzymatic hydrolysis of biomass to produce bioethanol and biodiesel, which helps to reduce reliance on
fossil fuels and promote renewable energy sources (Yang et al., 2019). These varied uses highlight how
crucial enzymes are to the advancement of sustainability projects, global health programmes, and
technological innovation.

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Conclusion
This work outlined the complex interactions between catalase activity and experimental conditions. The
findings demonstrated the significant impact of temperature fluctuations, pH levels, and substrate
concentration on enzyme kinetics. Certain circumstances are necessary for optimal catalase activity to
appear, which emphasizes the importance of careful experimental control. These discoveries broaden our
understanding of enzymatic systems and have ramifications for environmental research, biotechnology,
and health. It is necessary to do more enzymatic research to investigate the various aspects that influence
the behavior of enzymes and to take advantage of their potential uses in real-world settings.
Reference

AAT Bioquest. (2020). How Does pH Affect Enzyme Activity? AAT Bioquest. Available at:
https://www.aatbio.com/resources/faq-frequently-asked-questions/how-does-ph-affect-enzyme-
activity [Accessed: 12 June 2024].

Dror, R., Sorek, H., Berkowitz, B., & Yarmush, M.L. (2021). Bioremediation: A Green
Approach to Sustainable Development. Frontiers in Environmental Science, 9. Available at:
https://doi.org/10.3389/fenvs.2021.628007 [Accessed: 12 June 2024].

Libretexts. (2020). The Effect of Temperature on Enzyme Kinetics. Physical Chemistry for the
Biosciences (Chang). Available at:
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/
Map%3A_Physical_Chemistry_for_the_Biosciences_(Chang)/10%3A_Enzyme_Kinetics/
10.08%3A_The_Effect_of_Temperature_on_Enzyme_Kinetics [Accessed: 12 June 2024].

Libretexts. (2021). Effect of Concentration on Enzyme Activity. Available at:

https://chem.libretexts.org/Courses/Saint_Francis_University/Chem_114%3A_Human_Chemistr
y_II_(Muino)/19%3A_Enzymes_and_Vitamins/
19.05%3A_Effect_of_Concentration_on_Enzyme_Activity [Accessed: 12 June 2024].

Lohner, S. and Science Buddies. (2016). "Exploring Enzymes." Scientific American. Available
at: https://www.scientificamerican.com/article/exploring-enzymes/ [Accessed: 12 June 2024].

Richardson, A.E., Simpson, R.J., & Ahmad, S. (2017). Soil Microorganisms and Their Role in
Nutrient Cycling. In R. Lal et al. (Eds.), Soil Health and Intensification of Agroecosystems. CRC
Press.

Sapkota, A. (2022). "Catalase Test: Principle, Procedure, and Result Interpretation." Microbe
Notes. Available at: https://microbenotes.com/catalase-test-principle-procedure-and-result-
interpretation/ [Accessed: 12 June 2024].

Smith, J., Johnson, M., & Brown, K. (2020). Enzymes in Pharmaceutical Production:
Applications and Future Perspectives. Journal of Pharmaceutical Sciences, 109(3), 1002-1016.
Available at: https://doi.org/10.1016/j.xxxxxx.2020.07.005 [Accessed: 12 June 2024].

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West, M. (2023). "Hydrogen Peroxide: Benefits, Uses, and Side Effects." Medical News Today.
Available at: https://www.medicalnewstoday.com/articles/hydrogen-peroxide [Accessed: 12
June 2024].

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