LABORATORY REPORT

FOR

MIC180
SEMESTER OCT 2021 – FEB 2022

LAB 1: Effect of β-amylase on starch

NAME ADEENA FITRISHA BINTI ROSMAN

STUDENT ID 2021151741

GROUP AS1142A2

DATE OF SUBMISSION 12/11/2021

MARKS
Objective, title and /4
Introduction
Experimental /2
procedure
Results: /4
data, figures, graphs,
tables, etc.
Discussion, /6
conclusion &
references
Post lab questions /4
Total /20
TITLE
EFFECT OF β-AMYLASE ON STARCH

OBJECTIVE
To observe the progress of β-amylase-starch reduction reaction from t0 to t60.

INTRODUCTION
β-amylase is an enzyme that can hydrolyse starch at the α (1→4) glycosidic linkages
in polysaccharides to produce maltose units from the non-reducing ends of the chains.
This enzyme can also act in glycogen, starch, polysaccharides and oligosaccharides
producing beta-maltose by an inversion. During the ripening of a fruit, β-amylase
breaks starch into maltose, resulting in the sweet flavour of ripe fruit. It also presents
in an inactive form prior to seed germination. β-amylase plays a central role in the
complete degradation of starch to metabolizable or fermentable sugars during the
germination or malting of cereal grains. It also finds considerable application, together
with starch debranching enzymes in the production of high maltose syrups. It also
usually measured using a non-specific reducing sugar assays with starch as substrate.
Maltose also known malt sugar, is a disaccharide formed from two units of glucose
joined with an α (1→4) bond, formed from condensation reaction. Maltose is the
disaccharide produced when amylase breaks down starch. It is also can be found in
germinating seeds such as barley as they break down their starch stores to use for
food. It is also produced when glucose is caramelized.
3,5-Dinitrosalicylic acid is an aromatic compound that reacts with reducing sugars and
other reducing molecules to form 3-amino-5-dinitrosalicylic acid, which is strongly
absorbs light at 540nm. The reagent shows a differential behaviour towards mono and
disaccharides.
Spectrophotometer is a device used to measure the intensity of light as a function of
the colour of light and measures the amount of light absorbed by a sample.
Spectrophotometer techniques are mostly used to measure the concentration of
solutes in solution by measuring the amount of the light that is absorbed by the solution
in a cuvette placed in the spectrophotometer. The spectrophotometer technique is to
measure light intensity as a function of wavelength. It does this by diffracting the light
beam into a spectrum of wavelengths, detecting the intensities with a charge-coupled
device, and displaying the results as a graph on the detector and the on the display
device.
APPARATUS
Test tubes Cuvette
Beaker Measuring cylinder
Dropper Water bath
Micro pipette Spectrophotometer

MATERIALS
β-amylase Ice water
Acetate buffer 1.0 M, pH 5.0 Starch 1.0%
Distilled water DNSA reagents

PROCEDURE
1. The following reagent was mixed in a boiling tube and incubated at 30°C:

Material Volume (ml)

Starch 1% 4.0
Acetate buffer, 1.0 M, pH 5.0 4.0
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 was prepared.
4. The tubes at tblank, t0, t2, t4, t8, t15, t30 and t60 was labelled and kept on ice.
1.0 ml of the reaction mixture was added into DNSA tube labelled tblank.
5. To start the enzymatic reaction, 1.0 ml of β-amylase was added to the reaction
mixture and mixed it.
6. Immediately pipette 1.0 ml of the reaction mixture into the DNSA tube labelled
t0.
7. The 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.
8. 1.0 ml of water was added further to each DNSA tube to develop the colour and
read the absorbance at 560nm by using a spectrophotometer. tblank was used
to zero your spectrophotometer.
9. A graph of absorbance against time was plotted to see the progress of β-
amylase- starch reduction reaction.
RESULTS

Example results of spectrophotometer

DISCUSSION
Amylase is an enzyme that involved in starch breakdown and the bonding that involve
in starch breakdown are 1,4 glycosidic bonds. It catalyses the breakdown of starch.
When amylase reacts with starch, it cuts off the disaccharide maltose. As the reaction
progresses, less starch will be present and maltose will be present. At high
temperatures, Amylase becomes denatured, denatured amylase no longer catalyses
the hydrolysis of starch into glucose. 540 nm wavelength have been used because it
is the maximum wavelength that absorbed colour in highest developed using DNSA
reagent. Hence, we also need 60 minutes for effect starch degradation because at 0
to 5 minutes, the starch was not degraded yet since the amylase was just started to
act on the starch molecule and the rate should be lower. Then, after 5 to 40 minutes,
the reaction rate was increased once amylase enzyme degraded the entire starch
molecule into glucose or maltose.

CONCLUSION
In conclusion, the purpose of conducting this experiment is to see the effect of β-
amylase on starch. Based on the experiment, the reaction started when the DNSA
reagent added and β-amylase was mixed. The more concentration of amylase, the
greater effect it will have on the speed of enzyme activity. Therefore, β-amylase
catalyses the hydrolysis of the second α (1→4) glycosidic bonds, cleaving off maltose
at a time. At the end of the product, β-amylase will produce a maltose.

POST LAB QUESTIONS

1. What is the purpose of conducting this experiment?
To observe the progress of β-amylase starch reduction reaction from t0 to t60

2. Name the reducing sugar produced from the effect of β-amylase towards starch.
Maltose

3. Explain the flow of chemical process from the action of β-amylase towards
starch to the reaction of DNSA reagent towards the reducing sugar produced in
this experiment.
β-amylase breaks down starch to maltose by the process of hydrolysis. Maltose is a
disaccharide which is a reducing group that will break into a glucan. 3,5-
Dinitrosalicylic acid is an aromatic compound that reacts with reducing sugars and
other reducing molecules to form 3-amino-5-dinitrosalicylic acid, which is strongly
absorbs light at 540nm. The reducing group will reduce DNSA reagent that will cause
in colour changes from yellow to brown. The higher concentration of amylase, the
lighter the colour of the starch will appear.
 4. Draw the expected graph of absorbance against time to be observed in this
experiment.

REFERENCES
1. Siti Nur Husnina (2018, December 8), The effect of β-amylase on starch.
(Retrieved from https://www.youtube.com/watch?v=r8KYmLJh-II)

2. Lidiya Syahirah (2017, November 20), BCM202- Effect of beta-amylase on

starch (Retrieved from https://www.youtube.com/watch?v=4k0QZhT7hyU)

3. Megazyme (2019), (Retrieved from

https://www.megazyme.com/documents/Assay_Protocol/K-BETA3_DATA.pdf)

4. UKessays (2021, July 28), Starch hydrolysis of amylase

(Retrieved from https://www.ukessays.com/essays/biology/starch-hydrolysis-of-
amylase-biology-essay.php)