TITLE

Effect of β-amylase on starch.

OBJECTIVE

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

INTRODUCTION

Amylase is a digestive enzyme that is typically released by the pancreas and salivary
glands, while it is also very minimally present in other tissues. One of the earliest
enzymes in human history to receive scientific study, amylase was initially described in
the early 1800s. In the early 20th century, it was called amylase from the original name
diastase. The primary function of amylases is to hydrolyze the glycosidic linkages in
molecules of starch, turning them to simple sugars. There are three primary kinds of
amylase which are alpha, beta, and gamma amylase. They each affect the
carbohydrate molecule in a different way. In this experiment, we will be focusing on
beta-amylase which is also known as β-amylase.

Yeasts, moulds, bacteria, and plants, particularly the seeds, all contain
beta-amylases. They make up the majority of a combination known as diastase, which
is utilised to remove starchy sizing factors from textiles and transform cereal grains into
fermentable sugars. Maltose is created from starch by β-amylase through the hydrolysis
of α-1,4-glucan bonds. The main sources of β-amylase are sweet potatoes and higher
plant seeds. Due to the hydrolysis of starch into maltose during fruit ripening, β-amylase
is in charge of the sweetness of ripened fruit. The pH range between 4.0 and 5.5 is ideal
for β-amylase hydrolytic action. Many applications in science and industry have been
found for β-amylase. It is used to study the structural characteristics of molecules of
starch and glycogen generated in various ways.
 Starch, on the other hand, is a polysaccharide made up of glucose monomers
that are linked together by α-1,4-glucan bonds. The linear polymer amylose is the most
basic type of starch, and amylopectin is the branched type. In order to provide the plant
with a reserve food supply, starch is synthesised in the green leaves of plants using
extra glucose produced during photosynthesis. Granular forms of starch are stored in
chloroplasts and other plant storage structures. When necessary, starch is broken down
into its constituent monomer glucose units in the presence of certain enzymes and
water, which diffuse from the cell to nourish the plant tissues. Starch from plants is
broken down into its component sugar molecules in humans and other animals, which
subsequently provide the tissues with energy.

The spectrophotometer, which measures absorbance using monochromatic light

absorption, is the main instrument utilised. It is a tool that accurately monitors
electromagnetic energy at particular light wavelengths. It distinguishes between hues
and calculates how much of each colour is contained in a ray of light using the
properties of light and energy.

MATERIALS

APPARATUS MATERIALS

10 Test tubes 1 mL β-amylase

250 mL Beaker 5 mL Distilled Water

Measuring Cylinder 4 mL Starch 1%

8 Cuvette 4 mL Acetate buffer, 1.0 M, pH 5.0

Disposable Pipette 1 mL 2 mL DNSA reagent

Dropper Ice Water

Water Bath

Spectrophotometer
METHODS

1. The following reagents were mixed in a boiling tube and incubated at 30°C:

Material Volume (ml)

a) Starch 1% 4.0

b) Acetate buffer, 1.0 M, pH 5.0 4.0

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 was prepared.
4. The tubes were labelled at tblank, t0, t2, t4, t8, t15, t30 and t60. These tubes
were kept on ice. 1.0 ml of the reaction mixture was added into a DNSA tube
labelled tblank.
5. The enzymatic reaction was started by adding 1.0 ml of β-amylase to the reaction
mixture and mixed.
6. 1.0 ml of the reaction mixture was immediately pipetted into the DNSA tube
labelled t0.
7. 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 into each DNSA tube to develop the colour and the
absorbance was read at 560 nm by using a spectrophotometer. Tblank was used
to zero the spectrophotometer.
9. A graph of absorbance against time was plotted to see the progress of
β-amylase-starch reduction reaction.
RESULT

Diagram 1: Graph of absorbance against time

DISCUSSION

β-amylase will hydrolyse starch at the ɑ-1,4 linkage to produce maltose. Maltose is
disaccharide in which glucose is linked to glucose in which it possesses a reducing
group. Amylase is an enzyme that is involved in starch breakdown as well as 1,4 -
glycosidic bond. The DNSA method was used in this experiment to determine the
presence of reducing sugar of the free carbonyl (C=O) group of reducing sugars. This
involved aldehyde group and ketone group. DNSA turns into an orange colour under
alkaline conditions as the DNSA is reduced to 3 - amino,5 - nitrosalicylic acid.
 The standard curve of reducing sugar can be estimated with the optical density at
540 nm using a spectrophotometer. 540 mn is the maximum wavelength that absorbs
colour in the highest developed using DNSA reagent. We need 60 minutes or 1 hour for
the effect of starch degradation because in the beginning (at 0 - 4 minutes), starch was
not degraded yet since the amylase was just starting to act on starch molecules. Thus,
the rate should be lower. Next, after 4 - 30 minutes, the reaction rate was increasing
once the amylase enzyme degraded the entire starch molecule into glucose or maltose.
We are moving on after 30 - 60 minutes, as the reaction rate was slowing down since
that starch was limited and no more substrate in the sample available for the amylase
enzyme.

However, the result and data that we obtained are slightly different from the
expected as the absorbance data keeps fluctuating and unstable. Therefore, some
errors may occur during the experiment such as if β-amylase was not well shaken
before transfer into the test tube it will affect the final result of absorbance. Next, the
turbidity that is caused by thumbprint, where light cannot pass through the sample in
cuvette. Lastly, do not place the cuvette at the right arrangement in the spectrometer.
The precautions in this experiment are we have to make sure that there are no
thumbprints to avoid turbidity while handling the cuvette. Next, we have to make sure
that the cuvette is placed according to the sequence of orders and last but not least,
always place the β-amylase in a water bath to prevent the formation of precipitate.

CONCLUSION

The objective of this experiment is to observe the progress of β-amylase-starch

reduction reaction from t0 to t60. From the observations that have been made and the
result that we have obtained, we can conclude that this experiment was marked as a
failure since there are a few errors that happen during the experiment. The objective
was not obtained.
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.

The name of the reducing sugar produced from the effect of β-amylase towards
starch is 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.

The second α-1,4-glycosidic bond in starch is hydrolyzed by β-amylase, starting

from the non-reducing end, releasing two glucose units, or maltose, at a time.
Maltose is the reducing sugar created in this experiment. Light at 540 nm is
significantly absorbed by 3-amino-5-nitrosalicylic acid, which is created when
DNSA and maltose combine. The DNSA reagent will be reduced by the reducing
sugar, changing its colour from orange to dark orange.
4. Draw the expected graph of absorbance against time to be observed in this
experiment.

REFERENCE

1. Amylase - StatPearls - NCBI bookshelf. (2021, July 31). National Center for
Biotechnology Information.
https://www.ncbi.nlm.nih.gov/books/NBK557738/
2. HZDG. (n.d.). Spectrophotometer. Color Measurement Spectrophotometer
Supplier & Manufacturer.
https://www.hunterlab.com/blog/what-is-spectrophotometer/#
3. Starch. (n.d.). Encyclopedia Britannica.
https://www.britannica.com/science/starch