The Effect of pH and Temperature on the Rate of Hydrolysis of Starch
To investigate the effect of pH and temperature on the rate of
hydrolysis of starch using the enzyme diastase.
To be able to investigate the effects of the above aim, the theory of
how enzymes work and what affects them needs to be first looked into:
What Are Enzymes?
Enzymes are protein molecules, which can be defined as biological
catalysts. A catalyst is a molecule that speeds up a chemical
reaction, but remains unchanged at the end of the reaction. Enzymes
are globular proteins , which is a tertiary structure.
Individual amino acids
are joined together by peptide bonds by
condensation reactions forming a linear chain (see Fig 1). This is
called a primary structure.
It is maintained by covalent bonds between adjacent amino acids. A
polypeptide chain often coils into a a-helix due to hydrogen bonds
between neighbouring C=O and N=O groups. This is a secondary
structure. The secondary structure itself can be coiled or folded. The
shape of the molecule is very precise and held in this exact shape by
bonds between amino acids in different parts of the chain. Hydrogen
bonds, disulphide bonds between the cysteine molecules (which is an
amino acids that contains sulphur, C3H7NO2S), ionic bonds between
ionised amine and carboxylic groups and van der Waals interactions
between non-polar side chains keeping the tertiary structure in place.
The globular protein is a tertiary structure.
Globular proteins usually curl up so that their non-polar, hydrophobic
(water hating) R groups point into the centre of the molecule. The
polar, hydrophilic (water loving) R groups remain on the outside
making the molecule soluble.
Enzymes are specific; the tertiary structure gives a specific shape to
the active site so that only the substrate can fit. Therefore if the
bonds are broken within an enzyme the shape will alter not allowing
the substrate to fit.
Mechanism of Enzyme Action - Lock and Key
This explains the great specificity of enzymes for substrates and also
why changes in the enzymes three-dimensional shape cause alterations
to enzyme activity. [IMAGE]Fig 2
Factors Affecting Enzyme Controlled Reactions
Effect of Temperature
Initially the more heat that is given, the faster the molecules move
thus increasing the number of collisions that have sufficient energy
to bring about the reaction. However above a certain temperature the
structure of the enzyme molecule vibrates so energetically that some
of the bonds holding the enzyme molecule in its precise shape begin to
break and the enzyme is said to be denatured. When this happens it is
most unlikely that the three-dimensional shape will reform on cooling.
[IMAGE] Fig 3
The effect of temperature on the rate of a reaction can be expressed
as the temperature coefficient Q10.
Q10 = rate of reaction at (x + 100C)
Rate of reaction at x
Over a range of 0-40 0C, Q10 for an enzyme-controlled reaction is 2,
meaning the reaction rate is doubled for every rise of 10 0C . The
temperature that promotes maximum activity is referred to as the
optimum temperature. If the temperature is increased above this level,
then a decrease in the rate of reaction occurs despite the increasing
frequency of collisions, as explained above. However if temperature is
reduced to near or below freezing point, enzymes are inactivated, not
denatured. They will regain their catalytic influence when higher
temperatures are restored.
Effect of pH
Enzymes are sensitive to changes in pH. They contain groups that can
act as acids and bases, donating or accepting protons, when the pH
changes. This can change the number of ionic bonds and hydrogen bonds
present and therefore affect their three-dimensional arrangement.
Acidic amino acids have carboxyl functional groups in their side
chains. Basic amino acids have amine functional groups in their side
chains. If the state of ionisation of amino acids in a protein is
altered then the ionic bonds that help to determine the 3-D shape of
the protein can be altered. This can lead to altered protein
recognition or an enzyme might become inactive. A pH that is very
different from the optimum pH can cause denaturation. The optimum pH
value for most enzymes lies between pH 5 and pH 9. pH can have an
effect of the state of ionisation of acidic or basic amino acids.
As substrate concentration increases, the initial rate of reaction
increases. The more substrate molecules there are around, the more
often an enzymes active site is working. If more substrate is added,
the enzyme cannot work any faster.
[IMAGE] Fig 5
Figure 6 shows that the initial rate of reaction increases linearly.
In these conditions, the reaction rate is directly proportional to the
There are two sorts of enzyme inhibition:
1. Reversible - the activity of the enzyme is restored when the
inhibitor is removed.
* Competitive Inhibition - the inhibitor becomes attached to the
active site so that the substrate cannot bind.
* Non-competitive Inhibitor - this is still reversible but
increasing the ratio of substrate molecules cannot reduce the
degree of inhibition. The inhibitor becomes attached to the
enzyme, not the active site. The enzyme and/or the substrate
become changed and the enzyme activity stops.
2. Non-reversible Inhibitors - they bind firmly to the active site
N.B Iodine affects the reaction between starch
and diastase. Thus no
iodine will be placed in the reacting mixture.
Most enzymes have been found to contain non-protein groups, which are
essential for catalytic activity. In regards to this reaction between
starch and diastase, sodium chloride is the cofactor needed.
This is an enzyme mixture common in seeds, which is responsible for
. The mixture contains amylases for conversion of
starch to maltose and maltase for conversion of maltose to glucose.
This will be used in the experiments to show the progress of the
reaction. Iodine reacts with starch to produce a black/ blue colour.
This colour indicates the presence of starch. Thus when there is no
positive starch test it shows that it has been broken down by the
From the research several points can be made:
* It is important that no inhibitors are present in the reacting
mixture between starch and diastase, as this will affect the
results on the effect of pH and temperature.
* The pH also needs to be maintained so a buffer will be used in all
* As it has been shown that if the pH and temperature is are too
extreme then the reaction will not occur. Thus in the trial
experiments extreme values will not be used. With pH, not above 13
or below 2. With temperature not above boiling or below freezing.
These are counted as being extreme.
* Enzyme concentration must be kept at a constant for all
experiments and same volumes used.
* Substrate concentration must be kept at a constant for all
experiments and same volumes used.
Before a practical experiment can be carried out, the factors of the
reaction which when changed might affect the rate of the reaction,
must be identified. These factors are known as the key variables, and
deciding which to vary, and which to keep constant during the
experiment is important.
The following, are all the factors that should be considered when
investigating the rate of this reaction:
* The concentration of diastase
* The concentration of starch
* The pH of the buffer
* The temperature of the reaction mixture
Before any laboratory procedures are undertaken potential hazards need
to be identified.
* Enzymes are potential allergens and should be handled so as to
minimise contact or inhalation. They can be irritants. In case of
eye contact, immediately flush eyes with plenty of water for at
least 15 minutes. Seek medical attention if irritation develops or
* All substances must be in clearly labelled bottles.
* Lab Coat and Goggles to be worn AT ALL TIMES during the experiment
* Sodium Chloride solution causes eye irritation, so avoid contact
with eyes and wash hands after handling. In case of eye contact,
immediately flush eyes with plenty of water for at least 15
minutes. Seek medical attention if irritation develops or
* As glassware will be used, the up most care to prevent breakage
will be taken. This includes never placing cylindrical glassware
on the bench where it is able to roll off. If glassware is broken
then it should be cleared using appropriate equipment.
* Iodine solution is toxic and an irritant. Avoid contact with eyes.
Avoid direct contact with skin. Only use small quantities at any
time. In case of eye or skin contact, immediately rinse with
plenty of water for at least 15 minutes. Seek medical attention if
irritation develops or persists.
These will be conducted in order to identify any possible problems and
to find the approximate level of enzyme activity at different pH and
temperature values so an appropriate range of temperatures and pH
values can be used.
In this initial trial experiment, the aim is firstly, to become
acquainted with the method and procedure of the experiment; and
secondly, to confirm the fact that the pH affects the rate of the
In these trial experiments, the following apparatus will be used:
· 0.2M NaCl
· Test tubes
· 2% starch solution (fresh
· volumetric flasks
· 0.001M iodine solution
· Graduated pipettes
· Stop clock
· Spotting Tiles
· Thermostatically controlled water bath
Trial Experiment for Enzyme Activity on pH
5cm3 of 1% starch was put into 5 test tubes along with 1cm3 of 0.2M of
sodium chloride that was acting as a cofactor. Then 2cm3 of buffer
solutions was placed into each test tube. In each test tube a
different buffer was used, with pH's of 4.5, 5.9, 7.0, 8.0, and 8.8.
Then using a dropping pipette, 5 drops of 0.001M of iodine solution
were placed in each of the hollows in a spotting tile. Using a
graduated pipette 1cm3 of the diastase enzyme was added to each test
tube and immediately shaken. Then as soon as this had been done 3
drops of the reacting mixture in each test tube was placed in the
spotting tile with the iodine. From then on 3 drops was taken out
every two minutes, until there was no positive starch test or if there
was still a positive starch test after 10 minutes. Table 1 shows the
results of the first trial experiment. The, x, indicates a positive
starch test while, -, indicates no positive starch test.
pH Of Buffer
From these initial results it seems that the enzyme works best at a
lower pH. Thus the same experiment was done as above but with buffers
of lower pH values. Table 2 shows the results.
PH Of Buffer
Graph 1 shows the results of the trial experiment with pH against
Conclusions From Trial Experiment of pH
* In the main experiment pH's 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,
6.5, 7.0, 7.5, and 8.0. This range of pHs will give a good set of
results to plot a graph.
* In order to be able to get an accurate rate of reaction the sample
will be tested every 30 seconds rather than every 2 minutes, as in
the trial experiments.
* To ensure accuracy of the pH of the buffer, a pH meter will be
used. Graduated pipettes will be used to measure out all the
volumes, which should again increase the accuracy and avoid
* The test tubes in which the reaction is taking place in, will be
kept at room temperature to ensure that the only variable being
manipulated is that of pH
Trial Experiment For Enzyme Activity on Temperature
5cm3 of 1% starch was put into 5 test tubes along with 1cm3 of 0.2M of
sodium chloride that was acting as a cofactor. Then 2cm3 of a buffer
solution at pH 7 was placed into each test tube. Then each test tube
was placed at different temperatures at degrees Celsius at 10, 20, 30,
40, 50, 60, and 70. This was done using a water bath with the higher
temperatures and test tubes were placed in a beaker of ice cubes to be
able to reach the lower temperatures. The temperature was monitored by
placing thermometers in the test tubes. Then using a dropping pipette,
5 drops of 0.001M of iodine solution were placed in each of the
hollows in a spotting tile. Using a graduated pipette 1cm3 of the
diastase enzyme was added to each test tube and immediately shaken.
Then as soon as this had been done 3 drops of the reacting mixture in
each test tube was placed in the spotting tile with the iodine. From
then on 3 drops was taken out every two minutes, until there was no
positive starch test or if there was still a positive starch test
after 10 minutes. Table 3 shows the results of the first trial
experiment. The, x, indicates a positive starch test while, -,
indicates no positive starch test.
( 0C )
Conclusions From Trial Experiment of Temperature
* It can be seen that the rate of reaction between 30-500C is
similar. Thus readings will be taken every ten seconds rather than
every two minutes as in the trial and the rate will be measured
every five degrees at these points due to the sensitivity of
temperature on the rate.
* A buffer of pH 7 will be used in the main experiment because if
the optimum pH was used the reaction would go too quickly. Thus a
higher pH will be used to slow the rate. The buffer ensures that
the pH is not changing and the temperature is the only variable
that is changed.
Main Experiment For Enzyme Activity on pH
From the research it can be predicted that there will be an optimum pH
for the enzyme, and this will produce the fastest rate. On either side
of this optimum pH, the rate will decrease the further away it is from
Buffer solutions of pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,
7.5, and 8.0 were made up. The accuracy of the buffers was tested
using a pH meter and altered if necessary. Then for each pH, three
test tubes were arranged and in each one was put the following, using
a graduated pipette: 2cm3 of the buffer, 5cm3 of 1% starch solution,
and 2cm3 of 0.2M of sodium chloride. Then using a dropping pipette, 5
drops of 0.001M iodine solution were placed in each of the hollows in
a spotting tile. Using a graduated pipette 1cm3 of the diastase enzyme
was added to each test tube and immediately shaken. Then as soon as
this had been done 3 drops of the reacting mixture was placed in the
spotting tile with the iodine using a dropping pipette. From then on 3
drops was taken out every 30 seconds, until there was no positive
starch test or if there was still no positive test after 15 minutes.
The reacting mixture was kept constant at room temperature. At every
pH the experiment was repeated three times so that error bars could be
plotted on a graph.
The results of the experiments are shown in table 4. It shows the time
taken for no positive starch test to be shown. The results are plotted
in graph 3.
All of the results gained from all of the experiments conducted will
now be presented in a series of tables and graphs. In each case,
through conducting a large number of experiments it has been possible
to gain Maximum, Minimum, and Average results, displaying the
occurrence of any anomalies, or inaccuracies in the results. Where
possible this has been shown in the graphs, in the form of error bars.
Main Experiment for Enzyme Activity on Temperature
From the research it can be predicted that as the temperature
increases the iodine will show no positive starch test quicker,
because of the sugar being produced. However after a certain
temperature the reaction will slow down and eventually stop as the
enzyme will become denatured.
The rate was measured at temperatures of 0, 10, 20, 30, 35, 40, 45,
50, 60 and 70 degrees Celsius. Three experiments were taken at each
temperature in a test tube, so error bars could be plotted on a graph.
In each test tube the following was put in, using a graduated pipette:
5cm3 of 1% starch, 1cm3 of 0.2M of sodium and 2cm3 of a buffer
solution at pH 7. the test tubes were then placed at the desired
temperature using a water bath for the higher temperatures and test
tubes were placed in a beaker of ice cubes to be able to reach the
lower temperatures. The temperature was monitored by placing
thermometers in the test tubes. The diastase was then placed in a
separate test tube so it could reach the same temperature. Then using
a dropping pipette, 5 drops of 0.001M of iodine solution were placed
in each of the hollows in a spotting tile. Using a graduated pipette
1cm3 of the diastase enzyme was added to the test tube and immediately
shaken. Then as soon as this had been done 3 drops of the reacting
mixture in each test tube was placed in the spotting tile with the
iodine. From then on 3 drops was taken out every two minutes, with the
temperature continually being monitored. This was stopped if there was
still no positive starch test after 15 minutes of the reaction. Table
5 shows the results of the main experiment. The results are plotted in
Temp ( oC )
Analysis And Conclusions of Results
Now that all of the experiments have been conducted, the results
gained that have been displayed in the form of tables and graphs can
now be analysed. It may now be possible to draw certain conclusions
from the results about the nature of the reaction that has been
1. The Effect of pH on Enzyme Activity
From graph 3, which shows the rate (min-1) against pH, it seems that
the enzyme diastase works best between pH's 4 - 5.at pH 4.5 the
average rate was 0.010. The graph shows that as the rate of reaction
increases, the size of the error bars also increases. However the
shape of the graph does fit in with that of the predicted graph, as
shown in the initial research (Figure 4). There were a couple
anomalous results. For pH 5 the rate seemed to be too fast, at 0.011,
compared to the other results and pH 5.5 was too low. However, it
depends on the curve on the graph that was drawn. The actual process
of drawing graphs can provide an error. Drawing an accurate curve
freehand is quite difficult requiring a steady and smooth action, and
although this skill can be improved with practise, it still provides
sources of error. At pH 8, the reaction did not go too completion.
Graph 1, which was the trial experiment of pH is fairly similar too
that of graph 3. However, at pH 3 in the trial experiment the reaction
did not finish, unlike in the main experiment.
In conclusion, it would seem that the optimum pH is quite acidic,
between 4 - 5, and the enzyme stops working at pH 8.
2. The Effect of pH on Enzyme Activity
From the results it seems that the enzyme works best at around 40 - 45°C.
The shape of the graph is similar to the expected shape as shown in
Figure 3. However, there were a couple of anomalous results that were
missed out from the curve. The rate at 10°C and at 50°C were missed
Q 10 Rule
The results gained from investigating how the temperature affects the
rate of the reaction can now also be used to test the Q 10 rule.
The table below showing just a selection of results gained gives a
mixed response to the generally accepted rule that as there is a 10°C
rise in temperature, the reaction time is halved. Although from 0 - 10°C,
the rule seems to work from 10 - 20°C it is clear to see the rule has
Average Reaction Time
Now that the experiment has been conducted and the results have been
analysed, the investigation can be evaluated. By assessing methodology
and results, and identifying both errors and their sources.
Firstly, the reliability of the results should be assessed; can these
results be relied upon to give conclusions that show the true
patterns, or trends that actually occur in this reaction? It seems
that there is no reason to doubt the methodology behind this
investigation, the results gained show what was required to see how pH
and temperature affect the rate of reaction. The results in general
were consistent to what was expected. Whether the results compiled
show exactly what was happening when the experiments were being
conducted, is another question because there may be some doubt about
the accuracy of the results.
After assessing the methods and procedures used throughout this
investigation, areas where errors may have occurred have been
identified, and this may explain some of the uncertainty experienced
when analysing the results. In the results, some of the error bars
were quite large. This shows that the accuracy for these reactions was
not particularly high.
Firstly, the various solutions that were being used were not all taken
from the same batch of solutions. Due to the allotment of laboratory
time, it was impossible to use the same batches of solutions
throughout the whole experiment. This was because during the periods
when experiments were not being conducted, it was possible that the
solutions that were being used may have 'gone off', and therefore new
batches had to be made up. Therefore, each time a new batch was made
up, to say that it was exactly the same concentration as the previous
batch would be impossible. And so this is a very real area where
errors in the results may have occurred. Modifications that could be
made to increase the accuracy would be to only make up one large batch
of solutions, and conduct all of the experiments in one go, taking up
no more time than perhaps 48 hours. Along with this because many
people used the same solutions, due to practical reasons it is
possible that they may have become contaminated. If some of the starch
had been contaminated with the enzyme for example this would cause
problems and account for anomalies in the results.
Secondly, during the preparations of each experiment, the solutions
were measured out using graduated pipettes that measured to the
nearest 0.1cm3 only. Therefore there is a possibility that volumes
were not always measured to the accuracy capable, this may have been
due to bad technique, or possibly the fact that there was limited time
and a certain amount of pressure to complete all of the practical
work. Modifications that could be made to perhaps increase the
accuracy of the volumes measured could include using more accurate
pipettes; spending more time on both practising the technique, and
conducting the actual experiment.
When using the buffers, if left over a long period of time it was
found that the pH had actually changed. This could have been due to
oxidation of the solutions. However, because of time constraints it
was not practical to constantly make new buffers. Thus this can
account for errors in the results, as the pH of the buffer may have
been different to what was originally measured out. Thus to increase
the accuracy, new buffers should have been made up every day, using an
accurate pH meter.
Deciphering exactly when the reaction was complete was not always as
clear as expected, and this may account for any possible errors. The
nature of deciding when the reaction had finished was entirely
subjective, and may have changed from day to day, thus brining in
error. For a number of the experiments conducted, the colour change
that indicates when the reaction has completed, was not always as
instantaneous as previously described. On these occasions, the colour
change was relatively slow, and this made it difficult to determine
when exactly the whole of the solution had changed colour. Therefore
there may have been some variation in actually deciding the end point
of the reaction, which may have lead to errors and inaccuracies in the
results. One possible way to bring in a more objective test would be
to use Benedict's reagent as this would show when sugars were
produced. However, this would have been impractical considering the
timescale allowed and it does involve a colour change, which again
brings in subjectivity
When the ways in which temperature affected the rate of reaction was
investigated, it was decided to use a thermostatically controlled
water bath. Although this was much more accurate than using a bunsen
to heat the water, the accuracy of the water bath to maintain the
desired temperature is questionable. The water bath that was being
used seemed to be temperamental and not always particularly accurate,
this may have lead to errors in the results. Therefore a modification
that might be made could be to perhaps use a more sophisticated and
reliable water bath. The temperature couldn't be adjusted so the
reaction was occurring at the precise desired temperature; it was
Thus, as shown above there are numerous possibilities where error may
have occurred. These areas of error must therefore be used to explain
why the results appeared as they did, making it difficult draw
Products of the Hydrolysis of Starch
To find out the products when starch is hydrolysed by the enzyme
Starch can be considered to be a condensation polymer of glucose.
Starch may be highly branched (amylopectin) or relatively unbranched
(amylose). Starch is a polysaccharide. This is made up of a series of
monosaccharides, glucose, linked by 1µ-4 linkages. Each pair of
glucose units forms a maltose unit, as shown in figure 7.
It is already known from the research that diastase hydrolyses starch.
It contains amylases for conversion of starch to maltose and maltase
for conversion of maltose to glucose. Thus it can be predicted that
the starch hydrolysed by diastase will contain both maltose and
Iodine solution and Benedict's solution can both tell the difference
between starch and reducing sugars. It cannot however tell what the
reducing sugar is or if there are more than one type. Chromatography
however can distinguish each compound, by separating chemicals
according to their Relative Molecular Mass.
Identification of the products can be achieved in one of two ways:
1. The Rf value of each solute is calculated and compared to published
values in the same solvent. (Rf values are less than zero and have no
Rf = Distance moved by the solute
Distance moved by the solvent
2. A number of known substances are run in the same solvent as the
unknown substance, and the final positions of each are compared.
· Diphenylamine - Toxic. Possible mutagen. Harmful in contact with
skin, and if swallowed or inhaled. Irritant.
· Phenylamine - is toxic and harmful by skin absorption
· Phosphoric acid - Corrosive, causes burns. Harmful if swallowed and
in contact with skin. May be harmful through inhalation. Very
destructive of mucous membranes, respiratory tract, eyes and skin.
Paper Chromatography Method
A piece of chromatography paper was cut to about 25cm3 in length and
placed on a clean surface. To avoid contamination, the paper was held
at the top and plastic gloves were worn throughout the whole
experiment. A pencil line was drawn three centimetres from the bottom
of the paper and then four marks lightly along the line at four
centimetre intervals. At each mark a different letter was written. G
represented a sample of glucose, M represented a sample of maltose, E
represented a sample of starch that had been hydrolysed by the
diastase and A represented a sample formed by hydrolysing starch with
acid. Using a micropipette, the samples were taken and a small amount
was lightly dotted on the corresponding letter on each pencil mark.
The chromatography paper was then placed in a glass jar in a solvent
containing a mixture of propan-2-ol, ethanoic acid and water in the
ratio 3:1:1. A cover was then placed over the top and the chromatogram
was left to run, until it had reached ¾ of the way up. A locating
agent was then prepared to show up the spots. This consisted of 25cm3
of 2% phenylamine in propanone, 25cm3 of 2% diphenylamine in
propanone, and 5cm3 of 85% phosphoric acid. The paper was then drawn
through the locating agent in a shallow dish and then put in the oven
From Fig 8, the following can be said. The highest level reached is
that of glucose because it is the smallest molecule. Therefore, G and
A contain only glucose. This is acceptable with the research made. M
contains only maltose and is the lower than that of glucose because it
is a smaller molecule. E, which is the products of the hydrolysis of
starch by diastase, has two lines showing it contains both glucose and
Fig 8 - chromatography paper from experiment
Rf of G = 2.6/4.7 = 0.55 Rf of A = 3.2/5.5 = 0.58
Rf of M = 1.0/4.3 = 0.23 Rf of E = 1.2/4.4 = 0.27
= 3.5/5.0 = 0.70
This shows that G, A and E all contain glucose, even though the Rf of
E, being 0.70 is higher than the others, it can still be counted as
being glucose. Experimental error can account for this, as the drops
of liquids were not all at the same starting point on the
It also shows that M and E contain maltose.
From the results it can be said that the products of the starch
hydrolysis by diastase contain both maltose and glucose, which is in
line with the prediction made.
'Chemistry Students Book' - Nuffield Advanced Science
'Chemistry In Context' - Graham Hill And John Holman