The Effect of pH on Fungal and Bacterial Amylase
Amylase is a digestive enzyme systematized as a saccharidase (an
enzyme that splits polysaccharides). It is mainly a composition of
pancreatic juices and saliva, needed in order to break down long chain
carbohydrate insoluble molecules (such as starch) into smaller soluble
Enzymes are biological catalysts that speed up specific chemical
reactions and are made up of globular proteins made and used by our
bodies in human use. Enzymes are not only used in human natural
processes they are also used in manufactured and industrialised areas
such as in the making of bread, cheese, yoghurt, wine making etc. They
are used in various forms
of chemical reactions at temperatures
suitable for in living organisms of between 4 and 40 C . Enzymes are
vital for life as their deficiency in a reaction would cause very slow
reactions to maintain life.
The chemical(s) which an enzyme works on is called the substrate. An
enzyme combines with the substrate to form a temporary
enzyme-substrate complex. The higher the amount of enzyme to
substrate increases the chances of reactions. When the reaction occurs
the substrate is broken down into products and yet the enzyme does not
get used up and is free to work its cycle again.
There are certain factors which affect the rate at which these enzymes
can work and these are temperature, substrate concentration, enzyme
concentration and pH.
PH is a scale of 1-14 in which it contains chemicals which are acids
and alkalis. It is scale which is determines the strength of the acid
or alkali (pH 1 being strongest acid, pH 14 being strongest alkali and
pH 7 being neutral). Acids are chemicals between pH 1-6 which donate
H+ ions in aqueous solution and alkalis take away OH- ions away in
Initially enzyme amylases breakdown starches into substances that can
be absorbed by the body for use. Ptyalin is the name given to the
amylase present in saliva where the digestion of starch happens first
in the mouth.
The function of amylase enzymes are to catalyse and hydrolyse of 1,4
and 1,6 glycosidic linkages of polysaccharides (amylose and amyl
pectin) into a variety of products including maltose, dextrin and
D-glucose etc. Amylases are obtained from animal, bacterial, fungal
and plant sources such as barley, malt and fungi called Aspergillus
oryzae. There are a few forms of amylases.
Eukaryotes are membrane bound nuclei cells with some membrane bound
organelles. They carry out the processes of life. They are classified
as animals, plants, protoctists and fungi. Fungi are a huge group of
organisms that are ranged in size from unicellular yeasts to large
toadstools and puffballs etc and are found in a range of habitats;
aquatic and terrestrial. They are moulds that grow on dead damp
organic matter such as bread, non living vegetation etc.
Prokaryotes are non membrane bound cells and do not contain nuclei
and have a few of the organelles found in eukaryotes. They are
commonly made up of organisms such as bacteria. These bacteria species
are the smallest and oldest form of organisms with a cellular
structure. They come in a range of sizes but are much smaller than
eukaryotes. They live in environments of soil, air, dust, water and on
animals and plants.
My hypothesis is that:
‘The greater the pH the higher the activity of bacterial amylase
starch hydrolysis than fungal amylase‘.
Fungi and bacteria are both forms of amylases and are used in feeding
As mentioned above bacteria are prokaryotes they are very small
organisms with no true nucleus and a unicellular form and occur by
clumping together in characteristic patterns forming chains but the
cells can also be individual cells and have to be seen under
microscopes as they are very small. They can be found in most
environments such as dust, air, soil, water and living materials.
Together with fungi they play an important role in vital activities
with organisms in decaying of recycling of nutrients from organic
matter. Some bacteria can inhibit in many environmental extremities
of pH 1-11 in order to maintain constant cytoplasmic pH ranges.
Fungi are eukaryotes which are much bigger cells with membrane bound
nuclei and organelles. They compose of mildews, rusts, smuts,
puffballs, mushrooms, yeasts, stinkhorns, toadstools etc. They grow on
dead matter, parasites or dissolved nutrients through their cell
walls. It has a fungal body made up mycelium with parts of hyphae(
fine, thin, long threads) which form even longer branching chains of
fungi cells. Fungi usually grow best at slightly acidic pH of around
5-7 and so can reproduce at this range.
I have chosen bacterial amylase that work best at higher pH values
because they can be found in a range of different habitats and if this
is so it can tolerate the extremities in pH and so would have a larger
optimum pH range.
Generally any enzyme works best and faster at a certain optimum pH
range, under constant temperature. Some work in more acidic conditions
such as pepsin in the stomach will need to work best at pH 2 as it is
working with hydrochloric acid, and others in alkaline conditions.
Increase in temperature creates a bigger kinetic movement across the
molecules and so having a greater chance of them colliding with each
other. However enzymes also work at optimum temperatures before they
become inactivated (denatured). If one is working with pH the
temperature has to be kept constant as it is a factor that can also
The optimum pH is the maximum rate of reaction that occurs in the
range. When pH is altered below or above the optimum the activity is
decreased or becomes denatured. As pH is reduced it becomes acidic and
an increase in H+ ions which increase the amount of positive charges.
Alterations in pH change the ionic charges of the acidic and basic
groups and therefore mutate the R-groups in the amino acids which
disrupt the ionic bonding which help stabilise the specific shape of
the enzyme. The pH change therefore leads to the alteration of the
enzyme shape and also the active sites in which substrates are broken
down, causing the enzyme to denature.
[IMAGE] A graph showing enzyme activity against pH
I am going to investigate the effect of pH of the enzyme activity of
fungal and bacterial amylases using various pH and using it to
hydrolyse starch and testing it using potassium iodine solution for
starch present under timed conditions with these two different
The presence of starch can be discovered using iodine dissolved in
potassium iodide which is a orangey brown colour and changes to a dark
blue-black colour when starch is present. The experiment is to
investigate the effect of pH on the two types of amylases on its
activity. The relative activity of the amylases is noted by the time
taken for the starch substrate to be broken down resulting in a colour
that is no longer a blue- black colour when tested with the iodine
solution. This is known as the achromatic point.
In order for the experiments to be fair and reliable each time the
following must be taken in consideration:
starch concentration and volume will be the same in all experiments.
Amylase concentration and volume will be the same in all experiments.
The same concentration and drop size of iodine will be used on the
spotting time to determine starch presence.
Buffer solutions would be used to avoid any changes in pH differences
The iodine solution would be used as a guide to determine the end
Other volumes of liquid would measured accurately using measuring
cylinder and pipettes.
All this would be timed using a stop watch
0.1% fungal amylase and bacterial amylase
Potassium iodine solution
1.0% starch solution
White spotting tile
10 clean test tubes
Test tube rack
Buffer solutions ranging from pH 4-8
Take a test tube rack and place five clean test tubes each for the
fungal amylase and bacterial amylase and place a sticker with a
different pH of 4-8 so you do not get confused.
Prepare the buffer solutions by diluting the acid or alkali of certain
concentration (mol dm ) with 100ml of water. Use a universal indicator
to check the pH.
Pipette 1cm of either fungal or bacterial amylase decide which one
you are going to do first in each of the five test tubes. Then take a
pH solution of 1 cm and place in one of the test tubes with its
correct label. Time this for ten minutes. Whilst waiting place equal
drops of potassium iodine solution in different places on the spotting
tile. After ten minutes, add 5 cm 1.0% starch solution.
Now for every thirty seconds for five minutes, pipette a bit of the
solution and place it on a clean spot of iodine solution and record
the results. + is dark blue-black, +/- if dark brown, and - is no
Repeat this with the other pH solutions and the other amylase enzyme.
Risk assessment/ Safety precautions
It is brought in powdered form and so can causes respiratory problems
if inhaled in a poorly ventilated room. It may cause risk of eye
damage if contact with eye. On skin contact can causes cracked skin or
To avoid this wearing eye protection and gloves to be safe.
Handle with care wash with water if comes in contact with skin.
Protect from eyes as it can be irritant. Avoid by wearing gloves and
Iodine in potassium iodine solution:
It can be harmful if inhaled, swallowed or absorbed by skin. If
absorbed by skin or comes in contact with eyes it can be irritant. It
causes gastrointestinal discomfort if swallowed or respiratory
problems if inhaled.
Washing spotting tiles thoroughly under running water and clean any
WEAR SUITABLE PROTECTION WHEN HANDLING CHEMICALS.
Starch has very little harm or risk
USE ALL SUBSTANCES FROM CLEARLY LABBELED CONTAINERS WITH LIDS .