Nursing Biochemistry

Laboratory Report

Enzymes Chemical Nature

and Specificity of Enzymes
Activity No. 9

Cuenco, Lara Faith O.

Dela Torre, Sherieca L.
Kamdon, Mohammad

Locker No.36; NurBio Lab C

1st Semester, SY 2019-20
Nursing Biochemistry

Laboratory Report

RATIONALE
Enzymes are biological molecules (proteins) that act as catalysts and help complex reactions occur
everywhere in life. Let's say you ate a piece of meat. Proteases would go to work and help break down the
peptide bonds between the amino acids.

The enzymes in your body help to perform very important tasks. These include building muscle,
destroying toxins, and breaking down food particles during digestion. An enzyme’s shape is tied to its
function. Heat, disease, or harsh chemical conditions can damage enzymes and change their shape. When
this happens, an enzyme doesn’t work anymore. This affects the body processes the enzyme helped
support. Enzymes are produced naturally in the body. (Roland)

Enzymes are essential for healthy digestion and a healthy body. They work with other chemicals
in the body, such as stomach acid and bile, to help break down food into molecules for a wide range of
bodily functions.

Objectives:

⚫ To determine the properties of enzyme

⚫ To determine the presence of enzyme in every test

⚫ To test the specificity of enzyme

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

DATA INTERPRETATION

Table No. 1 Test for Catalase

Test Reagents added Result

Biuret (Potato extract) 10% NaOh4, 1% CuSO4 Turned to violet color

Discussion:

The Biuret Test is often used to determine the presence of peptide bonds in protein, and it detects the
presence of proteins in a sample. The test relies on a color change to confirm the presence of proteins.
The bonds present in biuret reagent are blue but then, if it will react with proteins the color will turn to
violet. According to Arnarson A. (2019) potatoes are primarily composed of carbohydrates and contain
moderate amounts of protein and fiber. Since biuret test is used to determine proteins, therefore the potato
extract gave a violet color.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

Table No. 2 Test for Catalase

Test Reagents added Result

Catalase Activity (Potato extract) 3% H2O2, 0.5% Benzitine Formation of bubbles

Did not change color

Theoretical result: Blue to green

coloration when benzitine is added

Discussion:

During the heating process a formation of sustained gas bubbles appeared, this indicates the presence
of a catalase.

Enzymes in potatoes are a type of catalase. When potato extract is mixed with hydrogen peroxide
solution, enzymes from the potato extract provide a reaction pathway for decomposition reaction of
hydrogen peroxide compound. The reaction produced is a foam or bubble this is a result of catalase
converting hydrogen peroxide into water and oxygen, the bubbles are filled with this oxygen. The more
bubbles produced the faster catalase is carrying out this reaction, or the more catalase present.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

With further research done it is found that hydrogen peroxide is detected by the oxidation of
benzidine or tolidine in the presence of peroxidase. When benzidine is added to potato extract the entire
solution should acquire bluee-green color. We think that the blur-green color did not appear in our sample
because base on the papers of Main and Shinn when freshly prepared potato extract us used the color will
develop to its maximal intensity within 4 to 6 minutes and it depends on the amount of peroxide that the
solution obtains.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

Table No. 3 Test for Specificity of Enzyme Action

Time Interval, minutes Test Tube1- Starch Color with Iodine Test Tube2- Glycogen Color with
Iodine

5 Chocolate Brown Chocolate Brown

10 Coffee Brown Coffee Brown

15 Dark Brown Dark Brown

20 Brown Brown

25 Dark Brown Brown

30 Dark Brown Light Brown

35 Brown Brown

40 Brown Brown

45 Brown Brown

50 Dark Brown Light Brown

55 Dark Brown Brown

60 Brown Brown

Theoretical Result: Blue-black color then Theoretical Result: As time passes

as time passes by the color turns lighter there are still no changes

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

Discussion:

In test tube 1 although it did not turn into blue-black color but then it turned to dark brown. As time
passes by we observed that there are small particles or tiny buildup like solid form that has a color of
black.

The enzyme amylase acts on the substrate starch catalyzing its break down into simple sugars.
Amylase will not catalyze the break down of the complex sugar sucrose into simple sugars. Amylase in
the starch (straight chain) form helices where in iodine molecules assemble and forms blue-black color.
While for amylopectin (branched portion) in glycogen forms much shorter helices and when iodine is
added, iodine are unable to assemble and only forms orange/yellow color.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

ANSWERS TO QUESTIONS

1. What is the Chemical nature of enzyme?

ANSWER:

All known enzymes are proteins. They are high molecular weight compounds made up
principally of chains of amino acids linked together by peptide bonds.

Enzymes can be denatured and precipitated with salts, solvents and other reagents. They
have molecular weights ranging from 10,000 to 2,000,000. Many enzymes require the
presence of other compounds - cofactors - before their catalytic activity can be exerted.

2. What type of enzyme is catalase?

ANSWER:

Catalase is a intracellular enzyme that speeds the decomposition of hydrogen peroxide into
water and oxygen. In this reaction the produced oxygen gas can be collected and used as a way
of measuring the reaction rate.

The catalase enzyme is one of the most efficient enzymes known, since each enzyme can
perform nearly 800,000 catalytic events per second. The key catalase function is protecting cells
from hydrogen peroxide (H2O2) molecules by converting them to oxygen (O2) and water
(H2O). H2O2 can damage DNA.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

3. What is the specificity of salivary amylase?

ANSWER:

The specificity of an enzyme refers to the types of chemical bonds with which the enzyme reacts.
Each enzyme has a particular range of what it can and cannot react with as well as a range of
conditions under which it will either be active or inactive. Most enzymes have a very specific target
and will only react when a particular chemical or chemical bond is present. Others are highly reactive
and will produce chemical reactions with almost any substance.

4. What mechanism is involved in the hydrolysis of starch by salivary amylase?

ANSWER:

Hydrolysis of starch or oligosaccharides by mammalian amylases, in general, results in

maltose as the leaving group. The active site of these amylases harbors three aromatic residues
Trp59, Tyr62, and Tyr151, which provide stacking interactions to the bound glucose moieties.

5. What are the degradation products of the enzymatic hydrolysis of starch?

ANSWER:

Whenever starch (polysaccharides) molecules undergo hydrolysis, it forms either

monosaccharides, disaccharides or trisaccharides. The end products depends on the strength of
enzymes used and the common enzymes are, α-Amylase, which produces the disaccharide maltose
and the trisaccharide maltotriose.

6. Account for the addition of NaCl and phosphate buffer.

ANSWER:

Phosphate-buffered saline (abbreviated PBS) is a buffer solution commonly used in

biological research. It is a water-based salt solution containing disodium
hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and
potassium dihydrogen phosphate. The buffer helps to maintain a constant pH.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

CONCLUSION

Enzymes speed the rate of chemical reactions. A catalyst is a chemical involved in, but not consumed
in, a chemical reaction. Enzymes are proteins that catalyze biochemical reactions by lowering the activation
energy necessary to break the chemical bonds in reactants and form new chemical bonds in the products.
Catalysts bring reactants closer together in the appropriate orientation and weaken bonds, increasing the
reaction rate. Without enzymes, chemical reactions would occur too slowly to sustain life.

T he functionality of an enzyme is determined by the shape of the enzyme. The area in which bonds of
the reactant(s) are broken is known as the active site. The reactants of enzyme catalyzed reactions are called
substrates. The active site of an enzyme recognizes, confines, and orients the substrate in a particular
direction.

Enzymes are substrate specific, meaning that they catalyze only specific reactions. For example,
proteases (enzymes that break peptide bonds in proteins) will not work on starch (which is broken down by
the enzyme amylase). Notice that both of these enzymes end in the suffix -ase. This suffix indicates that a
molecule is an enzyme.

Environmental factors may affect the ability of enzymes to function. You will design a set of
experiments to examine the effects of temperature, pH, and substrate concentration on the ability of
enzymes to catalyze chemical reactions. In particular, you will be examining the effects of these
environmental factors on the ability of catalase to convert H2O2 into H2O and O2.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

REFERENCES

Andrew Radar Studios. (2018). Chem4kids. Retrieved from:

http://www.chem4kids.com/files/bio_enzymes.html

Arnarson, A. (2019, March 7). Retrieved from:

https://www.healthline.com/nutrition/foods/potatoes#nutrition

Bank, E. (n.d.). What does a biuret test mean in Biology. Retrieved from:
https://education.seattlepi.com/biuret-test-mean-biology-4659.html

Brilliant Biology Student. (2016). What is Biuret test for protein. Retrieved from:
http://brilliantbiologystudent.weebly.com/biuret-test-for-protein.html

Brilliant Biology Student.weekly.com. (n.d.) Retrieved from:

http://brilliantbiologystudent.weebly.com/effect-of-temperature.html

Biochemical and Biophysical Research Communication. (2001, April). Retrieved from:

https://www.researchgate.net/publication/11458465_The_Mechanism_of_Salivary_Amylase_Hydrolysis
_Role_of_Residues_at_Subsite_S2'Carlos, C. (2013, April 21). Act 3, Enzymes. Retreieved from:
https://www.slideshare.net/caitlinvillacarlos012/lab-discussion

Liu, X. (2017). Microbial Enzymes of use in industry. Retrieved from:

https://www.sciencedirect.com/topics/neuroscience/catalase

Luo, Y. (n.d.). Retrieved from:

http://chemteacher.chemeddl.org/joomla/index.php?option=com_garyscookbook&Itemid=0&fun
c=rpdf&id=9&fbclid=IwAR3dG-EP8UARvLtjHGQzlq2cw8jnCTVffiQQL2un4mHZ1bX9KZY
ZvP-cSWU

Matt. (2012, March 5). Easy enzyme experiment. Retrieved from:

http://practicalbio.blogspot.com/2012/03/easy-enzyme-experiment-potato-catalase.html

Nguyen, D. (2019). Characteristics of a catalase enzyme. Retreieved from:

https://sciencing.com/characteristics-catalase-enzyme-14627.html

Opardt, C. (2003). Iodine-Starch. Retrieved from:

http://chemistry.elmhurst.edu/vchembook/548starchiodine.html

Sampson, S. (2019, January 15). Retrieved from:

https://www.healthline.com/health/why-are-enzymes-important#what-affects-enzymes

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry

Laboratory Report

Shinn, L. (1993). The determination of hydrogen peroxide in bacteria cultures. Retrieved from:
http://www.jbc.org/content/128/2/417.full.pdf

Science Copany. (2019). Food chemistry Experiments. Retrieved from:

https://www.sciencecompany.com/Food-Chemistry-Experiments.aspx

Study.com. (2003). What is the specificity of saliva amelase. Retrieved from:

https://study.com/academy/answer/what-is-the-specificity-of-salivary-amylase.html

University of Birmingham. (2019). What can affect enzyme controlled reactions. Retrieved from:
https://www.birmingham.ac.uk/undergraduate/preparing-for-university/stem/Biology/stem-legacy-enzym
es.aspx

Vignesh, M. (march 15). Quora. Retrieved from:

https://www.quora.com/What-products-are-made-by-starch-hydrolysis

Warthington Biochemical Corporation. (2019). Retrieved from:

http://www.worthington-biochem.com/introBiochem/chemNature.html

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University