Saliva Report

O1.11.
2017
BSc (HONS) Biotechnology Yr1

BASIC CELL BIOLOGY (agr1048) by MR
RANGHOO SANMUKhiYA
Effect of ph on enzymatic activity
Student ID 1713124
Table of Content
Pg
Introduction 3
Methodology 4
Procedures 5
Results 6-7
Discussion 8
Question 8
Conclusion 9
Reference 9
Introduction
Amylase is an enzyme that catalyzes hydrolysis of starch into sugars, in the human body,
amylase exists in saliva and in the pancreas , in the panceas it’s best pH are around 6.7 and 7.
In saliva, it does best between 4.6 and 5.2. depending on the optimal pH, the pH factor could
speed up the rate of reaction or denature and change shape of the enzyme causing it to
completely lose its functions p literally means the power (or concentration) of hydrogen
atoms in a substance. the point where the enzyme is most active - is known as the optimum
pH. The hypothesis pH 6.6 the enzyme would work best but at strongly acidic and al kaline
solution the enzym,e would denature was at The relationship between enzyme activity and pH
always looks like this (the curve can be steeper, if the optimal range is small and broader if
the range is wider):
Aim: To determine the effect of optimal pH for amylase to digest starch

Methodology
Apparatus used Material used

Test tube Distilled water
100 ml Beaker Saliva
Glass stirring rod Iodine solution
50ml Volumetric flask 1% starch
Plastic bottle Phosphate buffers of different
ph
Incubator
Stopwatch
Procedures
1.a) Collection of saliva:
 The mouth was washed with water and saliva was collected by spitting into a 100ml
beaker until a volume of about 10ml was obtained. The saliva was used for the
following experiments.
b) Determination of chronic period
 5ml of saliva of saliva was diluted with 50ml distilled water in a beaker, mixed well
by stirring. This diluted saliva was used for the following experiments on chronic
period..
 In each one of a series of test tubes arranged in a rack, 2ml of distilled water was
pipette and 5 drops of 1/5 M iodine solution ( Tube A) was added.
 5ml of 1% starch was pipetted in a clean test-tube ( Tube B)
 1ml of 0.2 M phosphate buffer of a specific pH and and 1ml of 0.3 % NaCl to test
tube B . mixed.
 Tube B mixture was incubated in a water bath at 38ͦ C for 3minutes.Titration should
The total volume of NaOH used to titrate each sample were recorded.
 After exactly 1 min , 3 drops of the solution were transferred from the digestion test-
tube (Tube B) to the test-tube containing iodine ( Tube A)
 Similarly at intervals of 1 ml ,3 drops aliquots from Tube B to subsequent iodine
tubes.
 The time was noted when the blue black colour due to starch reacting with iodine
was no longer obtained.
 The above experiment was repeated with the two phosphate buffers of different Ph.
Results
For ph 10
Ph
Minutes 3.5 6.6 10

1 Blue black Brown Brown
2 brown Brown Blue black
3 brown Brown Blue starting to fade
4 brown Brown Orange
5 yellow Brown Yellow
6 yellow Brown Yellow
7 yellow brown Yellow
graph 1 shows rate of reaction against ph

10
9
8
7
6 graph 1 shows rate of
reaction against ph
5
4
3
2
1
0
3 4 5 6 7 8 9 10 11
Discussion
For this experiment blue black colour should be observed, when we put the constituent of
tube B in the iodine solution at the beginning. And the expected colour change was blue/black
then blue violet then red/violet then red/brown light brown and finally yellow. According to
the result shown in the table., ph 3.5 it took 5min for the amylase to digest the starch . pH
3.5 is acidic , the expected result was that at pH 3.5 it should be the last to change as it was
the most acidic. As , it would denature the enzymes. The hypothesis was that the more neutral
the pH , it is less likely to denature the enzyme. For ph 6.6 , amylase did not digest starch
anymore as no yellow colour was observed. The expected result was the hypothesis . For ph
10 it takes about 7 minutes to turn clear. The expected result were the pH of 10 would change
solution of iodine and 1% of starch first as it was more basic meaning would break down
starch faster. Therefore the product can easily fit in the amylase’s active site can easily,
Did our Hypothesis supported?
Our hypothesis states that at nearly neutral Ph the amylase would digest starch first that is at
a faster time, as at extreme acidic ph and extreme alkaline , the pH enzyme would denature.
But this practical showed that Ph 10 also seems to be good as at this Ph it turned yellow
before the almost neutral one pH 6.6 . so the hyphothesis was not supported.
Questions
1. Why NaCl is used?

It was used since the Cl- ions in it acts as a co- factor for ptyalin action and the buffer
maintains an optimum pH for enzyme action
2. Explain briefly what is meant by chromic point.
It is the point in time during the action of amylase on starch at which the reaction
mixture no longer gives a colour with iodine
Conclusion
To sum up, the ph 10 found to be the best condition for the digestion of starch.
References
i. http://www.nuffieldfoundation.org/practical-biology/investigating-effect-ph-amylase-
activity
ii. https://prezi.com/jwnkz0dxuuy5/the-effects-of-ph-on-amylase/

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O1.11.

BSc (HONS) Biotechnology Yr1

Effect of ph on enzymatic activity

Aim: To determine the effect of optimal pH for amylase to digest starch

Apparatus used Material used

1.a) Collection of saliva:

b) Determination of chronic period

Minutes 3.5 6.6 10

graph 1 shows rate of reaction against ph

Did our Hypothesis supported?

1. Why NaCl is used?

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