BIO462

BIOCHEMISTRY

LAB REPORT 3 : ENZYMOLOGY

PART 1: DETERMINATION OF
OPTIMUM PARAMETERS.

NAMES MATRIC NUMBERS

CORLAINE SHIERY ANAK JAMES 2023899958

FATIMAH BINTI HASRIN 2023491958

NURFARAH NABIHA BT AHMAD RASDAN 2023492024

NUR SHAZLIYANA BINTI HASRIN 2023638078

NUR FATIHAH BINTI MOHD RAKIDIN 2023414874

TITLE ENZYMOLOGY PART 1: DETERMINATION OF OPTIMUM
PARAMETERS.

INTRODUCTION Enzymes are a class of protein functioning as biological catalysts.

They control the chemical reactions that occur in a cell or
organism. The effect of this is the rate at which the chemical
reactions take place. Enzymes can cause the reactions to occur
thousands to a million times faster than they would without the
enzyme being present.

Enzyme names usually end in the suffix,-ase. This may be added

to the word of the chemical involved in the reaction such as
sucrase, an enzyme which works on the sugar, sucrose. Enzymes
are also very specific in controlling chemical reactions. Each
chemical reaction that takes place in a cell is controlled by different
enzymes. This means that a cell contains hundreds to many
thousands of different enzymes. The enzyme that will be used in
this lab is called amylase.

Amylase is an enzyme that breaks down into sugar. Amylase is

present in human saliva, where it begins the chemical process of
digestion. The pancreas also makes amylase (alpha amylase) to
hydrolase 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.
all amylases are glycoside hydrolases and act on α-1,4-glycosidic
bonds.

OBJECTIVE(S) ● To establish the optimum parameters of enzyme amylase.

● To recognize the effect of enzyme concentration on the rate
 of reaction.
● To examine the effect of substrate concentration on the rate
of reaction.
● To observe the effect of pH on enzyme activity.

MATERIALS
Lab Activity 1- Calibration and Settings for
Spectrophotometer
● Spectrophotometer
● Calibration cuvette
● Starch in dropper bottle
● Distilled water
● Parafilm square

Lab Activity 2- The Effect of Enzyme Concentration on The

Rate of Reaction
● Spectrophotometer
● 4 cuvettes
● Starch in dropper bottle
● Amylase
● Dropper (for enzyme mixture)
● Distilled water
● Marking pen
● Plastic ruler
● Parafilm squares
● Data Collections sheet

Lab Activity 3- The Effect of Substrate Concentration on The

Rate of Reaction
● Spectrophotometer
● 7 cuvettes
● Starch in dropper bottle
● Amylase
● Dropper
 ● Distilled water
● Marking pen
● Plastic ruler
● Parafilm squares
● Data collection sheet

Lab Activity 4 – The Effect of pH on Enzyme Reaction

● Spectrophotometer
● 6 cuvettes
● pH buffers 3,5,7,9 and 11
● Starch in dropper bottle
● Amylase
● Dropper
● Distilled water
● Marking pen
● Parafilm square
● Spreadsheet

PROCEDURES
Lab Activity 1- Calibration and Settings for
Spectrophotometer
i.The spectrophotometer was turned on and let it warm 5 minutes
ii.Prepare the calibration cuvette:
(a) 2 cm from the bottom of the cuvette wad marked.The mark was
placed on the side of the cuvette so it will not interfere with the light
beam of the spectrophotometer.
(b)Cuvette is then filled with distilled water to the 2 cm mark.
(c)10 drops of starch was added,placing the parafilm over the
opening of the cuvette and hold it in place with your finger.The
contents was mixed by inverting the tube several times.
iii.The spectrophotometer was set to 410 nm.
iv.The spectrophotometer was set to Absorbance (A).
v.Chamber cover was opened.
vi.The calibration cuvette was inserted into the holder.
vii.Closed the chamber cover.
viii.The absorbance was read and recorded.
ix.The calibration cuvette and its contents was saved.

Lab Activity 2- The Effect of Enzyme Concentration on The

Rate of Reaction

i.Four cuvettes are numbered as 1-4.

ii.2 cm from the bottom of the cuvette is measured up using a ruler
and marked using a marking pen. It is done for all four cuvettes.
iii.Each cuvette is then filled with distilled water to the 2 cm mark.
iv.Preparation of cuvette contents:
(a)5 drops of enzymes were added to cuvette 2,15 drops to
cuvette 3 and 45 drops to cuvette 4.
(b)10 drops of starch are added to each of the 4 tubes.
(c)Cuvettes were left for 5 minutes.
v.Cuvette 1 is placed in the spectrophotometer at the end of minute
5 and its absorbance was measured with the previously calibrated
spectrophotometer.
vi.Step 5 was repeated with cuvettes 2,3 and 4.
vii.Results were recorded.
viii.Cuvette’s content is then emptied into the waste container
provided.
ix.Cuvettes were rinsed out and dried.

Lab Activity 3- The Effect of Substrate Concentration on The

Rate of Reaction

i. Seven cuvettes are numbered as 1-7.

ii. 2 cm from the bottom of the cuvette is measured up using a ruler
and marked using a marking pen. It is done for all seven cuvettes.
iii. Each cuvette is then filled with distilled water to the 2 cm mark.
iv. The cuvette contents are prepared with:
a) 1 drop of starch is added to cuvette 1, 3 drops to cuvette 2, 5
drops to cuvette 3, 10 drops to cuvette 4, 20 drops to cuvette 5, 40
drops to cuvette 6 and 60 drops to cuvette 7.
b) Then, 10 drops of enzymes are added to each of the 7 cuvettes.
c) Cuvettes were let to set for 10 minutes.
d) The tubes are aerated over the 10 minutes with the cuvettes
covered with parafilm and shaking them.
v. Cuvette 1 is placed in the spectrophotometer at the end of
minute 10 and its absorbance is measured.
vi. Step 5 is repeated with cuvettes 2-7.
vii. Results are recorded.
viii. Cuvette’s content is then emptied into a waste container
provided and it is rinsed out and left to dry.

Lab Activity 4 – The Effect of pH on Enzyme Reaction

i. Six cuvettes are numbered as 1-6.

ii. 2 cm from the bottom of the cuvette is measured using a ruler
and marked using a marking pen. It is done for all six cuvettes.
iii. The cuvette contents are prepared with:
a) Cuvette 1 is filled up to the 2 cm mark with pH3 buffer solution
and 10 drops of enzyme.
b) Cuvette 2 is filled up to the 2 cm mark with pH5 buffer solution
and 10 drops of enzyme.
c) Cuvette 3 is filled up to the 2 cm mark with pH7 buffer solution
and 10 drops of enzyme.
d) Cuvette 4 is filled up to the 2 cm mark with pH9 buffer solution
and 10 drops of enzyme.
e) Cuvette 5 is filled up to the 2 cm mark with pH11 buffer solution
and 10 drops of enzyme.
f) Cuvette 6 is filled up to the 2 cm mark with distilled water and 10
 drops of enzyme.
g) Then, 10 drops of starch are added to each of the 6 cuvettes.
h) Cuvettes are left to stand for 5 minutes.
i) The tubes are aerated over the 5 minutes with the cuvettes
covered with parafilm and shaking them.
iv. Cuvette 1 is then placed in the spectrophotometer at the end of
minute 5 and its absorbance is measured with the previously
calibrated spectrophotometer.
v. Step 4 is repeated with cuvettes 2,3,4,5 and 6.
vi. Results are recorded.
vii. Cuvette’s content is then emptied into the waste container
provided.
viii. Cuvettes are rinsed out and dried.

RESULTS

BLANK 1 2 3 MEAN

0.894 0.890 0.892 0.892

Table 1: The absorbance of blank at 410 nm

A. ENZYME CONCENTRATION

CUVETTE OD 1 0D 2 OD 3 MEAN

1 1.004 1.003 1.005 1.004

2 0.846 0.841 0.834 0.840

3 0.311 0.304 0.291 0.302

4 0.165 0.164 0.164 0.1643

Table 2: The absorbance of different enzyme concentration in

 each cuvette

B. SUBSTRATE CONCENTRATION

CUVETTE OD 1 OD 2 OD 3 MEAN

1 0.129 0.130 0.132 0.130

2 0.127 0.131 0.131 0.130

3 0.176 0.182 0.183 0.180

4 0.317 0.296 0.292 0.302

5 0.294 0.304 0.296 0.298

6 0.567 0.544 0.556 0.556

7 0.575 0.572 0.565 0.571

Table 4: The absorbance value of different substrate concentration

in each cuvette

C. pH

CUVETTE OD 1 OD 2 OD 3 MEAN

1 0.167 0.166 0.168 0.167

2 0.210 0.216 0.213 0.213

3 0.334 0.333 0.331 0.333

4 0.587 0.583 0.581 0.584

5 0.494 0.499 0.501 0.498

6 0.411 0.398 0.399 0.403

 BLANK = 0.89
Table 4: The absorbance value of different pH

DISCUSSION In this experiment, the rate of enzymatic processes is frequently

determined by measuring the absorbance of light at 410 nm
wavelength. The concentration of substrate and enzymes, as well
as pH, are important variables that affect how quickly these
reactions occur and, in turn, affect how much absorbance
changes. The principle of kinetic assay is that, if the concentration
of the substrate is sufficiently high in comparison to enzyme then
the rate of reaction will be proportional to the concentration of the
enzyme. Therefore, the amount of product formed in a given
period of time would be proportional to the amount of active
enzyme present, with all other factors remaining constant. (Dr.
Vivek Pant, 2022).

Table 2 shows that when the concentration of the enzyme

increases, the absorbance decreases. The rate of reaction and
absorbance should both rise proportionately to an increase in
enzyme concentration, according to theory. This suggests that
there are more accessible active sites for the substrate to bind to
and undergo a reaction. Despite additional increases in enzyme
concentration, there may come a moment at which the rate peaks,
meaning that all of the available active sites have already been
used in the reaction. Table 2 shows that the absorbance on the
first cuvette without amylase is 1.004, the highest value while the
value of absorbance with 5 drops of amylase is 1.004. Next, the
cuvettes containing 15 and 45 drops of amylase produced
absorbance values of 0.840 and 0.302 respectively. Figure 1
illustrates the inversely proportional graph that was generated as a
result. Thus, this demonstrates that our experimentally constructed
outcomes are at odds with theory.
According to Table 3, one drop of starch and three drops of starch
have the same absorbance at 0.130. Cuvettes 3 and 4 yielded
absorbance values of 0.180 and 0.302 with 5 and 10 drops of
starch, respectively. However, there is a slight decrease for the fifth
cuvette, with the absorbance dropping to 0.298. This data
indicates that an error has to be made when making the
concentrated substrate solution. The actual concentration
employed in the test could change due to variability in substrate
solution preparation, which would affect the response rate that is
measured. Next, using 40 and 60 drops of starch, respectively,
cuvettes 6 and 7 have absorbance values of 0.556 and 0.571.

With reference to Figure 2, we can see that the absorbance rose

sharply at first and gradually leveled out, creating a hyperbolic
curve, as the substrate concentration increased. This trend is in
line with Michaelis-Menten kinetics, which states that at greater
substrate concentrations, the enzyme's active sites become
saturated and the reaction rate reaches a plateau. The initial sharp
rise in absorbance with increasing substrate concentration
suggests that the enzyme molecules have plenty of accessible
active sites to bind with the substrate while the substrate
concentration is low. As a result, the enzyme-substrate complex
forms more quickly and produces more product, which is reflected
in higher absorbance measurements. However, the enzyme's
active sites gradually get saturated as the concentration of
substrate rises. At this moment, the rate of reaction reaches a
maximum (Vmax) and the majority of enzyme molecules are
already bind to substrates. Because of this, increasing the
substrate concentration further does not considerably speed up the
reaction, which causes the absorbance curve to plateau.

Lastly, the absorbance value at each pH in each cuvette was

shown in Table 4. There are increasing and diminishing tendencies
 in the pattern of the graph at Figure 3. Cuvettes one through four
exhibit a growing trend in absorbance, with pH values of 3, 5, 7,
and 9 correspondingly. The absorbance are 0.167, 0.213, 0.333
and 0.584 respectively. Meanwhile, the absorbance of the pH 11
buffer and distilled water solutions decreases to 0.498 and 0.403,
respectively, indicating a declining trend. Most enzyme activity
follows a bell-shaped curve, which decreases to zero in the strong
alkaline region and increases from zero in the strong acid region
up to a maximum value as shown in Figure 3. Two different effects
responsible for this behavior are the state of protonation of
functional groups of amino acids and cofactors involved in the
catalytic reaction and the native, three-dimensional protein
structure of the enzyme. (H. Bisswanger, 2014). The graft showed
that amylase functions best at a pH of 9, which is shown by the
greatest absorbance. In fact, the optimal pH of amylase is at the
pH 0f 7. There could be discrepancies in the particular enzyme
variant under study, methodological issues, or experimental
settings that differ from the generally acknowledged ideal pH for
amylase.

CONCLUSION In conclusion, the objectives of the experiment which is to

accurately calibrate the spectrophotometer, determine the enzyme
and substrate concentration on the rate reaction, respectively and
to observe the effect of pH on the enzyme activity were
achieved. It shows that there is a linear relationship between the
enzyme concentration and the rate of reaction. The graph on the
effect of substrate concentration on the rate of reaction shows that
it will yield a hyperbolic graph and as for the effect of the pHon the
enzyme activity, we managed to get the graph that is similar to the
theoretical graph curve. The pH has a major influence on the
enzyme amylase activity as each enzyme has their own
designated optimum pH where the rate of reaction of
the enzyme is the highest. All in all, it is substantial that alteration
 in enzyme and substrate concentration as well as the pH level do
affect the enzyme activity and the rate of the reaction.

REFERENCES Viven Pant. (2022, April 11). Importance of Observing the

Progress Curve During Enzyme Assay in an Automated
Clinical Chemistry Analyzer: A Case Study. The Journal of
the International Federation of Clinical Chemistry and
Laboratory Medicine Vol 33 (1): 56-62.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9092719/
Hans Bisswanger. (2014, March 21). Enzyme Assays.
Elsevier GmbH, Vol 1: 41-55.
https://doi.org/10.1016/j.pisc.2014.02.005