Investigating factors which affect the rate of the Amylase Enzyme in converting Starch to Maltose

150/ 101 = 1.49cm3

Again this is a difficult volume to measure out so I shall say that 2cm3 equals one part. This will again therefore allow me extra solution for spillages etc.

        Therefore:

1 x 2cm3 starch 100%         = 2cm3

100 x 2cm3 water                = 200cm3

In this way I can see that the total amount of amylase 100% needed is 8cm3. This will be made from a solid, where 1gcm-1 represents a 100% solution concentration. I therefore require 8g amylase and 2g starch for the temperature variable.

Despite the fact that I am making different quantities of amylase solution to starch solution I will always add them in the same quantity to keep the experiment fair and only have one variable.

Variable 2- Amylase Concentration

• Again the table above is a rough one showing the kind of data I hope to record.         Again there are 15 readings for each concentration. However because the amylase         concentrations are different I will have to make up each concentration separately.  

• 15 readings x 4cm3 (maximum cuvette volume) means I will need to make a total of 60cm3 solution for each concentration.

• As I am having 1cm3 amylase: 1cm3 starch, I will only require 30cm3 of each to make the combined total.

• Below is how I shall make each concentration:

Amylase 5% = 1 part amylase 100%: 20 parts water

Therefore each part will be:

        30 / 21 = 1.43cm3

Once again this is a difficult volume to measure so I will make extra solution and make one part equal to 2cm3. Therefore:

1 x 2cm3 amylase 100%         = 2cm3

20 x 2cm water                 = 40cm3

Amylase 4% = 1 part amylase 100%: 25 parts water

Therefore each part will be:

30 / 26 = 1.15cm3

This is also difficult to make so I shall make 1 part equal to 2cm3. Therefore:

1 x 2cm3 amylase 100%         = 2cm3 

25 x 2 cm3 water                 = 50cm3

Amylase 3% = 1 part amylase 100%: 33 parts water

Therefore each part will be:

30 / 34 = 0.88cm3

Once again this is an awkward volume to make so I shall make 1 part equal to 1cm3. Therefore:

1 x 1cm3 amylase 100%         = 1cm3

33 x 1cm3                         = 33cm3

Amylase 2% = 1 part amylase 100%: 50 parts water

Therefore each part will be:

30 / 51 = 0.59cm3

This is once again difficult to make accurately so I shall make one part equal 1cm3. Therefore:

1 x 1cm3 amylase 100%         = 1cm3

50 x 1cm3 water                 = 50cm3

Amylase 1% = 1 part amylase 100%: 100 parts water

Therefore each part will be:

30 / 101 = 0.30cm3

This is tricky to make accurately so I will instead make 1 part equal to 1cm3. Therefore:

1 x 1cm3 amylase 100%         = 1cm3

100 x 1cm3 water                 = 100cm3

As the reactants are being put in as 1cm3 amylase: 1cm3 starch I will require 180cm3 starch solution.

Therefore to make 1% starch solution I will need to have:

                1 part starch 100%: 100 parts water

Therefore I will have 2cm3 starch 100% and 200cm3 water. This will give me a little over the required amount.

Variable 3- Copper Sulphate Inhibitor

As you can see in the above table, the copper sulphate concentrations are very small. I have decided these volumes based on previous research, in which I investigated the action of copper sulphate on catalase. I therefore expect the reaction to occur somewhat the same, with small concentrations having large effects on the output of the enzyme.

        

Once again my standard solutions for this variable will be amylase 5% and starch 1%. As there are 90 readings in total and a cuvette volume of 4cm3, I will require about 360cm3 total solution. I therefore require approximately 180cm3 of amylase and 180cm3 of starch.

To make the 5% amylase solution:         1 part amylase 100%: 20 parts water

Therefore there are 26 parts in total so each part is:

180 / 21 = 8.6cm3        this is another awkward volume so I shall make one part equal to                                 9cm3.

Therefore:

1 x 9cm3 amylase 100%         = 9cm3

20 x 9cm3 water                 = 180cm3

As the reactants are being put in as 1cm3 amylase: 1cm3 starch I will require 180cm3 starch solution.

Therefore to make 1% starch solution I will need to have:

                1 part starch 100%: 100 parts water

Therefore I will have 2cm3 starch 100% and 200cm3 water. This will give me a little over the required amount.

As the copper sulphate concentrations are so small I will only require maybe 1cm3 of copper sulphate solution.

To make 0.005% copper sulphate concentration: 1 part

Variable 4- Starch Concentration

My 4th variable will be to see how the action of amylase is affected by substrate concentration. Below is a rough table showing some of the main details I will try and obtain.

The total solution required for each concentration is:

15 x 4cm3 (max cuvette volume) = 60cm3

As the enzyme and substrate and being added in equal quantities I will require 30cm3 starch solution and 30cm3 amylase solution for each concentration

Starch 5% = 1 part starch 100%: 20 parts water

Therefore each part will be:

        30 / 21 = 1.43cm3

Once again this is a difficult volume to measure so I will make extra solution and make one part equal to 2cm3. Therefore:

1 x 2cm3 starch 100%         = 2cm3

20 x 2cm water                 = 40cm3

Starch 4% = 1 part starch 100%: 25 parts water

Therefore each part will be:

30 / 26 = 1.15cm3

This is also difficult to make so I shall make 1 part equal to 2cm3. Therefore:

1 x 2cm3 starch 100%         = 2cm3 

25 x 2 cm3 water                 = 50cm3

Starch 3% = 1 part starch 100%: 33 parts water

Therefore each part will be:

30 / 34 = 0.88cm3

Once again this is an awkward volume to make so I shall make 1 part equal to 1cm3. Therefore:

1 x 1cm3 starch 100%         = 1cm3

33 x 1cm3 water                = 33cm3

Starch 2% = 1 part starch 100%: 50 parts water

Therefore each part will be:

30 / 51 = 0.59cm3

This is once again difficult to make accurately so I shall make one part equal 1cm3. Therefore:

1 x 1cm3 starch 100%         = 1cm3

50 x 1cm3 water                 = 50cm3

Starch 1% = 1 part starch 100%: 100 parts water

Therefore each part will be:

30 / 101 = 0.30cm3

This is tricky to make accurately so I will instead make 1 part equal to 1cm3. Therefore:

1 x 1cm3 starch 100%         = 1cm3

100 x 1cm3 water                 = 100cm3

As the reactants are being put in as 1cm3 amylase: 1cm3 starch I will require 180cm3 amylase 5% solution.

Therefore to make 5% amylase solution I will need to have:

                1 part amylase 100%: 20 parts water

Therefore I will have 9cm3 amylase 100% and 180cm3 water. This will give me a little over the required amount.

Practical Procedure

Temperature Variable

1. Make up the solutions into their concentrations as shown in the table below and do so in an appropriate sized beaker.

2. Place 10cm3 of the amylase solution into a test tube and another 10cm3 starch solution in another test tube. Then place both these test tubes into a water bath of 0*c.
3. Once both of the solutions are at the same temperature of 0*c pour the contents of one into another. At this point start the timer.
4. Once this is done, using a pipette, take a sample of the solution and place this into a cuvette. Then add 3 drops of Benedict’s solution and place the cuvette in the colorimeter. Note the time on the stop clock in doing, and make sure every time you put the next cuvette in that one minute has elapsed. This is to ensure the experiment is fair.
5. Record the light transmission through the cuvette.
6. When another minute has elapsed since taking a sample of the two solutions, repeat the above steps and record to see how the light transmission changes with time.
7. Do this for 5 minutes- by which time you should have 5 results.
8. Repeat experiment for the other temperatures, by setting the water bath to the temperature required.
9. Repeat the experiment twice more to account for any anomalies.

Amylase Concentration Variable

1. Make up the amylase and starch concentrations as shown in the table below. Make sure the beakers in which you are making the solutions are clean.

2. Place 10cm3 amylase 5% solution into a test tube and 10cm3 starch solution into another.
3. Add the two contents together and start the timer
4. Take a sample of the solution immediately after and place this into a cuvette.
5. Add 3 drops of Benedict’s solution
6. Note time and place the cuvette into the colorimeter
7. Record light transmission
8. When one minute has passed take another sample and repeat steps 5 to 7
9. After 5 minutes have passed move on to the next concentration and repeat the procedure
10. Carry out 2 more repeats to account for any anomalies

Copper Sulphate Inhibitor Variable

…………

Starch Concentration Variable

…………

Scientific Theory

Temperature Variable

        I think that changing the temperature will have an effect on the rate of the reaction of amylase on starch. Before I talk about the chemistry of the effect of temperature here is a little information about enzymes.

An enzyme can be defined as a biological catalyst and like any other it is affected by the conditions it is in. The enzyme has a specific shape and this means only substrates with a certain shape can be broken down by a certain enzyme. For this reason amylase can only break down starch, due to the substrates fitting into the active site. It can not however break down lipids due the lipid substrates having a structure which does not allow it to fit in the active site of the amylase enzyme.

        Amylase is a complex 3 dimensional globular protein. The active site, which is usually a cleft in structure, contains some amino acids which carry out the breakdown of a substance.

        H                R                O

        Diagram showing structure of a simple

                N        C        C                        amino acid

        H                                OH

The enzyme is the quaternary structure of a protein. It is held together by several bonds, which are hydrogen bonds, ionic bonds, disulphide bonds and hydrophobic interactions between non- polar side chains. This structure is brought about by the long amino acid chains coiling up on themselves. Hydrogen bonds then form between the –CO groups of one amino acid and the –NH group of another, which hold this shape in place. This is called an α- helix and is the secondary structure. This structure may coil up into a precise three- dimensional shape which is the tertiary structure.

The shape of the active site in an enzyme is determined by the R groups. The large variety of different R groups means different shape active sites can exist, explaining why the enzymes are specific to one substrate type.

The diagram below taken from ‘Biology 1’ illustrates how an enzyme works.        

Diagram showing how an enzyme speeds up the breakdown of a substrate

The above diagram relates to how the amylase enzyme in my reaction works. In the left diagram we can see that the enzyme and substrate are in a mixture. The substrate, which in my investigation is starch, moves into the active site of the enzyme. The two then bind and form an enzyme-substrate complex. It is held in place using temporary bonds which form between the R groups of the enzyme’s amino acid and the substrate. These bonds are weak and thus are not covalent

        The specific shape of the enzyme can be understood by the lock and key diagram. The substrate above fits the shape of the active site, so can bind with it. Any other shape will not fit this active site. This can be resembled by a lock and a key theory in that if the key, i.e. the substrate, is not the right shape it will not fit in the lock, which is the enzyme.  

Finally the interactions between the substrate and active site of the amylase enzyme cause the starch to break down. The temporary bonds which form during this process cause a higher tendency for the breakdown of a substance, which in turn reduced the activation energy.

Now that we know what an enzyme is we can see how altering the temperature would cause a change in the rate of the reaction.

For a reaction to occur the particles must collide with a certain minimum kinetic energy. The size of this kinetic energy needed varies between reactions due to different bond enthalpies. This minimum energy is known as the activation energy. The enthalpy profile of a reaction looks like the one below

In a solution like mine, which will contain amylase enzymes and starch, the particles have a range of different kinetic energy. Most of the particles will be moving at moderate speeds, others will have slightly greater kinetic energy and some will have slightly less. When the temperature of the reactants rises, they move around faster and have a greater amount of kinetic energy.

        This means that of those particles that collide with one another, the amount of energy of the impact is more likely to exceed the minimum kinetic energy required. The amylase enzyme works by lowering this minimum energy, or activation energy, further, so that a larger number of molecules have the required energy and can cause a reaction. This is illustrated in the diagram below.

The above diagram shows that only a small proportion of the molecules have the energy E to overcome the activation energy (which in this case is 50kg mol-1), and to cause a reaction to occur. If we now however raise the temperature the graph will look like the one shown below.

Distribution curves showing effect of a temperature rise of 10K on the proportion of reactions with greater than 50kg mol-1

From the graph you can see that by increasing the temperature by 10 Kelvins the graph has shifted to the right- i.e. there is a higher average kinetic energy of each particle. There is a much higher proportion of molecules with greater than 50kg mol-1 which means more collisions will be successful enough for a reaction to occur.

Yet to talk about effects of inhibitor and concentration variables.

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