Nursing Biochemistry

Laboratory Report

Enzymes
Factors Affecting Enzymes
Activity No. 10

Yap, Alain John E. (Principal Author)

Omar, Princess Marizhar M.
Sanaani, Nur Fatima S.
Locker No.40 NurBio Lab C
1st Semester, SY 2019-20
Nursing Biochemistry
Laboratory Report

RATIONALE
Enzymes are macromolecular biological catalysts that accelerate chemical reactions. The molecules upon
which enzymes may act are call substrates, and the enzyme converts the substrates into different
molecules known as products. Almost all metabolic processes in the cell need enzyme catalysis in order
to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze
individual steps. An enzyme is defined as a protein synthesized in the living cells, which catalyzes
thermodynamically possible reaction so that the rate of the reaction is compatible with the biochemical
processes needed for the maintenance of the cell.

DATA INTERPRETATION
Enzymes are biological molecules (typically proteins) that significantly speed up the rate of virtually all
of the chemical reactions that take place within cells.

They are vital for life and serve a wide range of important functions in the body, such as aiding in
digestion and Metabolism. The molecules that an enzyme works with are called substrates. The
substrates bind to a region on the enzyme called the active site. Enzymes are made up of amino
acids which are linked together via amide (peptide) bonds in a linear chain. This is the primary
structure. The resulting amino acid chain is called a polypeptide or protein. The specific order of amino
acids in the protein is encoded by the DNA sequence of the corresponding gene. (Joseph Castro, 2014)

General Structure of Enzyme Primary Protein Structure Secondary Protein Structure

Objectives:

o To be able to know the properties of an enzyme

o To be able to test the presence of an enzyme
o To be able to test the specificity of an enzyme

____________________________________________________________________________________________________ 1
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

A. Preparation of Catalase

Catalase acts as the catalyzing enzyme in the decomposition of hydrogen peroxide. Nearly all living
things possess catalase, including us! This enzyme, like many others, aids in the decomposition of
one substance into another. Catalase decomposes, or breaks down, hydrogen peroxide into water and
oxygen.

Solution REAGENTS ADDED RESULT

Violet
Biuret Test (on potato extract) 2 ml 10% sodium hydroxide

Catalase Activity (on potato Brown color

1 ml 3% hydrogen peroxide
extract)

 The Biuret Test is done to show the presence of peptide bonds, which are the basis for
the formation of proteins. These bonds will make the blue Biuret reagent
turn purple. And it is Positive

There’s a violet color formed on the biuret test on potato extract it indicate that there is a present of
protein in the Solution. The reagent used in the Biuret Test is a solution of copper sulfate (CuSO4) and
sodium hydroxide (NaOH).The NaOH is there to raise the pH of the solution to alkaline levels; the
crucial component is the copper II ion (Cu2+) from the CuSO4.When peptide bonds are present in
this alkaline solution, the Cu2+ions will form a coordination complex with 4 nitrogen atoms

Documentation:

____________________________________________________________________________________________________ 2
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

The Catalase Activity is done also instead of biuret to find out if there is a presence of proteins the
reagents used are 3% Sodium Peroxide and 0.5% benzidine and the result is in brown color therefore
there’s no protein presence in catalase activity and it is Negative

Documentation:

A. Specificity of Enzyme Action

One of the properties of enzymes that makes them so important as diagnostic and research tools is the
specificity they exhibit relative to the reactions they catalyze. A few enzymes exhibit absolute specificity;
that is, they will catalyze only one particular reaction. Other enzymes will be specific for a particular type
of chemical bond or functional group. In general, there are four distinct types of specificity:

Absolute specificity - the enzyme will catalyze only one reaction.

Group specificity - the enzyme will act only on molecules that have specific functional groups, such as
amino, phosphate and methyl groups.

Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of
the molecular structure.

Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.

Though enzymes exhibit great degrees of specificity, cofactors may serve many apoenzymes. For
example, nicotinamide adenine dinucleotide (NAD) is a coenzyme for a great number of dehydrogenase
reactions in which it acts as a hydrogen acceptor. Among them are the alcohol dehydrogenase, malate
dehydrogenase and lactate dehydrogenase reactions.

____________________________________________________________________________________________________ 3
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

Table No. 1 Temperature

Time Interval(min) Test Tube 1 10○C Test Tube 2 40○C Test Tube 3 60○C

5 Dark Brown Dark Blue to Black

10 Dark Black Brown Dark Brown

15 Dark Black Brown Dark Brown

20 Dark Black Brown Dark Brown

25 Dark Black Brown Dark Brown

30 Light Black Brown Dark Brown

35 Light Black Brown Dark Brown

40 Light Black Brown Light Brown

45 Clear Black Brown Light Brown

50 Clear Black Brown Light Brown

55 Clear Black Brown Light Brown

60 Clear Black Brown Light Brown

As the Time goes by the color change and turn to light No change at all.

Documentation:

ANSWERS TO QUESTIONS
1. What is the chemical nature of an enzymes?

____________________________________________________________________________________________________ 4
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

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.

2. What is the specificity of salivary amylase?

ANSWER: Amylase is an enzyme that catalyses the hydrolysis of starch (Latin amylum) into
sugars. Amylase is present in the saliva of humans and some other mammals, where it begins the
chemical process of digestion. Foods that contain large amounts of starch but little sugar, such as
rice and potatoes, may acquire a slightly sweet taste as they are chewed because amylase
degrades some of their starch into sugar. The pancreas and salivary gland make amylase (alpha
amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by
other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce
amylase. As diastase, amylase was the first enzyme to be discovered and isolated (by Anselme
Payen in 1833). Specific amylase proteins are designated by different Greek letters. All amylases
are glycoside hydrolases and act on α-1,4-glycosidic bonds. Salivary amylase is an enzyme that
can digest starch molecules and break them down to sugar molecules. Salivary amylase is an
enzyme that can digest starch molecules and break them down to sugar molecules.
3. 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.
We hypothesized that Tyr151, located at the S2' subsite, may influence the size of the leaving
group. Therefore, using a baculovirus expression system, we generated a mutant Y151M in which
the tyrosine at position 151 of human salivary amylase is replaced by a methionine. The specific
activity, K(m), rate of hydrolysis, and the product distribution for Y151M were distinctly
different from those of the wild-type enzyme using starch and oligosaccharides as substrates. The
mutant enzyme Y151M consistently produced glucose as the minimal leaving group and
exhibited a twofold increase in K(m). These results suggest that the stacking interaction at subsite
S2' in the wild type plays a role in hydrolysis.

____________________________________________________________________________________________________ 5
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

4. 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.
5. Why are NaCl and phosphate buffer added in the test of enzyme specificity?
ANSWER: The materials used in examining the activity of enzymes and specificity of enzymes
were the following: enzyme solution (2ml saliva + 18ml distilled H2O + 60ml 0.5% NaCl),
buffered starch (1% starch in phosphate buffer) – for temperature, 2% unbuffered starch – for pH.
Acetate buffer solutions (pH 4 and 5), Phosphate buffer solutions (pH 6.7 & 8), and bicarbonate
buffer (pH 10) were also used to examine the effect of pH with a water bath set to 37°C. Constant
temperature bath (4, room temp., 37, 50, 60, and 70°C) were used to examine the effect of
temperature on salivary amylase. Effect of Temperature.

CONCLUSION

The denaturation of an enzyme or the altering of its in reaction speed is affected by three main factors:
temperature, pH and the concentration of enzymes. Temperature is important to thereaction of an enzyme
because being too hot or cold can increase or decrease the rate. Also, being too hot, as seen in experiment
five can denature the enzyme so that is unable to function normally once put back into normal
circumstances. Due to this, the hypothesis for temperature was correct because it was said that a high
temperature would begin the process of denaturation, which was shown by the lack of color change in
tube E. The cold temperature hypothesis was also correct, because it was hypothesized that the enzyme
would slightly increase in color changeafter being cold, which it did as seen in tube A. The pH level of a
reaction cannot be too high or low because it can change the shape of the active site and inhibit the
process. The hypothesis for this was only partially correct because those who had a pH that was low did
not yield as dark of a color change as the neutral pH did, but the high pH did also yield some color
change. The concentration of an enzyme verses a a substrate can also increase the rate of the enzyme
reaction, as seen in experiment two and three where the higher concentrations yielded a darker color. If
this experiment were to be conducted again, it would be beneficial to create somekind of predetermined
color range because qualitative data is more difficult to give a number to and this causes changes in the
data based on a particular person’s perspective.

REFERENCES

https://www.britannica.com/science/protein/The-specificity-of-enzymes

____________________________________________________________________________________________________ 6
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

https://www.livescience.com/45145-how-do-enzymes-work.html

https://biology-igcse.weebly.com/food-test-4---biuret-test-for-proteins.html

https://www.slideshare.net/caitlinvillacarlos012/lab-discussion

____________________________________________________________________________________________________ 7
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University