LABORATORY REPORT 
FOR 
MIC180 
SEMESTER MAC 2023 – SEP 2023 
 
LAB 1: Effect of β-amylase on starch 
 
GROUP MEMBERS 1. AMIRUL IMRAN BIN AMIRUDDIN (2022838166)
NAMES  2. AHMAD FARID BIN MOHD NAZREE (2022846432)
3. MUHAMMAD NUR SYAZWAN BIN ZAINAL ABIDIN
(2022479748)
4. IMRAN AIMAN BIN IBRAHIM (2022816932)
5. MUHAMAD AKIMI BIN OTHMAN (2022878156)
6. MUHAMMAD SHAHIRRUDIN HAIKAL BIN AZURUL SHAH
(2022897456)
GROUP  AS1142C1 
DATE OF 11 April 2, 2023 
th

SUBMISSION 
 
 

MARKS   
Objective, title and   /4 
Introduction 
Experimental procedure  /2 
Results:  /4 
data, figures, graphs,
tables, etc. 
Discussion, conclusion /6 
& references 
Post lab questions  /4 
Total  /20 
LABORATORY 1: EFFECT OF β-AMYLASE ON STARCH
1. Objective
 To understand the role of enzymes in the breakdown of complex molecules
into simpler ones.
 To study the effect of Beta-Amylase on Starch.
 To observe the progress of β-amylase-starch reduction reaction from t0 to t60

2. Introduction
Enzymes are important proteins that use to catalyze biochemical reactions in living
organisms. They are responsible for speeding up chemical reactions that would or
else be too slow to sustain life. Enzymes are very specific due to their specific active
shapes. In another word, it is only intended to catalyze a specific reaction.
Enzyme specificity is determined by its three-dimensional shape, which is
determined by its amino acid sequence (The Editor Of Encyclopedia Britannica,
2023)
In this experiment, an enzyme β-amylase is used, which is in charge of breaking
down starch into maltose. In this experiment, β-amylase activity was measured by
monitoring the rate of starch hydrolysis using a colourimetric assay. This assay uses
a DNAA reagent that reacts with reducing sugars to produce a unique colour. The
rate of colour development is directly proportional to the concentration of reducing
sugars in the reaction mixture and proportional to the rate of starch hydrolysis by β-
amylase. (DNSA reagent base, n.d.)
This experiment aims to determine the effect of different factors, such as enzyme
concentration, pH, and temperature, on the rate of β-amylase-catalyzed starch
hydrolysis. The reaction progress curve will be plotted by measuring the absorbance
of the DNSA reagent at regular intervals, and the rate of reaction will be determined
by analyzing the slope of the curve.

3. Material
4.0 ml of Starch 1%, 4.0 ml of Acetate buffer, 1.0 M, pH 5.0 and 5.0 ml of
Distilled water.

4. Material
 polisterin box of the ice  Pipette filler
box  Cuvette
 4.0 ml of Starch 1%  Pipette
 4.0 ml of Acetate buffer,  Beaker
 1.0 M, pH 5.0  3,5 Di Nitro Salicylic Acid
 5.0 ml of Distilled water. (DNSA)
 Spectrophotometer  β-amylase
5. Method
1) The reagents were mixed in a boiling tube and incubated at 30°C.

Table 1 List of materials

Materials Volume
(ml)
a) Starch 1% 4.0
b) Acetate buffer, 1.0 M, 4.0
pH 5.0
c) Distilled water 5.0

2) β-amylase was placed in a separate tube and incubated at 30°C.

3) A series of labelled tubes containing 2.0 ml of DNSA reagent were prepared
and labelled at t blank, t0, t2, t4, t8, t15, t30, and t60. The tubes were kept
on ice, and 1.0 ml of the reaction mixture was added into the DNSA tube
labelled t blank.
4) To start the enzymatic reaction, 1.0 ml of β-amylase was added to the
reaction mixture, and it was mixed.
5) Immediately, 1.0 ml of the reaction mixture was pipetted into the DNSA tube
labelled t0.
6) Sampling was repeated at times 2, 4, 8, 15, 30, and 60 minutes by pipetting
1.0 ml of the reaction mixture into the appropriately labelled DNSA tube.
7) 1.0 ml of water was added to each DNSA tube to develop the colour, and
the absorbance was read at 560 nm using a spectrophotometer. t blank was
used to zero the spectrophotometer
8) A graph of absorbance against time was plotted to see the progress of the
β-amylase-starch reduction reaction.

6. Result

Graph Absorbance against Time

0.7
0.6
0.5
Absorbance

0.4
0.3
0.2
0.1
0
0 2 4 8 15 30
Time

Figure 2 Absorbance against Time graph

7. Discussion
This experiment looked into the enzymatic action of -amylase on starch. The
experiment involved combining a starch, acetate buffer, and distilled water
solution and incubating it at 30°C. The reaction was then started by adding
the enzyme -amylase to the reaction mixture, and samples were taken
throughout the set of time.

The results of the experiment show that the absorbance of the DNSA reagent
increases with time, indicating that the starch concentration decreases as the
enzyme β-amylase hydrolyzes the starch. A plot of absorbance against time
was made to show a steady decrease in starch concentration over the course
of the experiment.

The expected result of this experiment is to get the graph to show of

continuously increasing of absorbance along with the increase of time. This is
because the starch will continue to break down into maltose with the help of β-
amylase until all of it finish. When the process of breaking down end, the
graph will be constant until the end showing that there’s is nothing to be
broken down anymore or in another word is that the reaction is
complete.Maltose will have the same concentration as starch at the beginning
of the experiment.However, the result that we got from the experiment was
different from what the theory stated.

The possibility of contamination or measurement errors during the preparation

of the solutions is one potential source of error in this experiment. The results
dependability could also be impacted by the enzyme's possible denaturant or
degradation during storage or handling. Another possibility is the
spectrophotometer is having an instrumental error. A few ways to reduce error
is to repeat the record of the result by using another spectrophotometer.

8. Conclusion
The purpose of this experiment is to understand the role of enzymes in the
breakdown of complex molecules into simpler ones and study the effect of
Beta-Amylase on Starch along with observing the progress of the β-amylase-
starch reduction reaction from t0 to t60.

The limitations of this experiment are the spectrometer in producing results.

The error may have been caused by a misaligned optical path, a damaged
detector, or other issues related to instrument calibration. This also affects
graphing resulting in Figure 1.

In future experiments, it is important to use a properly functioning

spectrophotometer to ensure accurate measurements. Any inconsistency in
 data must be carefully investigated as the source of error and addressed to
ensure the result are accurate and reliable.

9. Question
1) What is the purpose of conducting this experiment?
to track the development of starch reduction by amylase from time t0 to time
t60.

2) Name the reducing sugar produced from the effect of beta-amylase

towards starch.

Maltose

3) Explain the chemical process flow from the action of β-amylase towards
starch to the reaction of DSNA reagent towards the reducing sugar in this
experiment.

Amylase digests starch into smaller molecules, ultimately yielding maltose,

which is cleaved into two glucose molecules by maltase. In this experiment,
we work with the enzyme β-amylase. This enzyme is responsible for
hydrolyzing starch from 3,5-dinitro salicylic acid turns to 3-amino,5-
nitrosalicylic acid. (Vendatu, 2023)

4) Draw the expected graph of absorbance against the time to be observed in

this experiment.

Absorbance agaisnt ti me
0.7
0.6
0.5
Absorbance

0.4
0.3
0.2
0.1
0
1
Time in minutes
10. References
DNSA reagent base. (n.d.). Retrieved from University Of Reading:
https://www.ncbe.reading.ac.uk/dnsa-reagent-base/#:~:text=DNSA%20is%20more
%20sensitive%20and,where%20reducing%20sugars%20are%20produced.

the editor of encyclopedia britannica. (2023). enzyme. Retrieved from britannica.

The Editor Of Encyclopedia Britannica. (2023, March 27). Enzyme. Retrieved from Britannica:
https://www.britannica.com/science/enzyme

Vendatu. (2023, April 7). Retrieved from Maltase - Enzyme:

https://www.vedantu.com/chemistry/maltase