Enzyme Investigation - Amylase

Ateeq Rashid

Enzyme Investigation – Amylase

Plan

I am going to investigate the effects of four different concentration of amylase solution on the breakdown of starch. I have chosen amylase because enzymes act on one particular substance or groups of substances i.e. they have a specific function. The shape of the molecule, especially the active site is linked with this specificity. A simple way of explaining this specificity is in terms of the lock and key hypotheses. The shape of the active site matches the shape of the substrate molecule so the substrate can fit into the active site and amylase can react and breakdown starch.

According to Susan Toole, ‘Advance Human and Social Biology’, ‘the active site of an enzyme may be used again and again. Enzymes therefore work efficiently at very low concentrations. The number of substrate molecules which an enzyme can act upon in a given time is called its turnover number. Provided the temperature and other conditions are suitable for the reaction, the rate of a reaction should be directly proportional to the enzyme concentration’ (page 35).

Figure 1 shows ‘the rate of reaction in the first 30 seconds for an enzyme concentration investigation’.

Graph taken from ‘Advance Science Biology 1, page 45’

I predict that as the concentration of amylase solution increases, the rate of reaction will also increase. This is because more active sites will be available as more enzyme molecules are present, therefore more substrate molecules will be broken down. The rate of reaction will be slower if a lower concentration of amylase solution is used; this is due to the fact that there would be a competition for the substrate to react with the active sites. I expect the rate of reaction to be directly proportional to the enzyme concentration as the more enzymes are present, therefore more active sites would be available for the substrates to slot into.

Apparatus

I used the following apparatus:

5 pipettes

1 white spotting tile

1 test tube rack

1 stop-clock

4 syringes

4 test tubes

Method

Using a pipette, place two drops of iodine solution into each cavity of the white spotting tile.
Using a 0-10cm syringe, empty 9cm of the starch solution into each of the four test tubes. Place the tubes in a rack.
Using a 0-1cm syringe, add 1cm of the 1% amylase solution to one of the four test tubes and mix the contents.
Immediately start the stop-clock and after withdrawing up ...

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Apparatus

I used the following apparatus:

5 pipettes

1 white spotting tile

1 test tube rack

1 stop-clock

4 syringes

4 test tubes

Method

Using a pipette, place two drops of iodine solution into each cavity of the white spotting tile.
Using a 0-10cm syringe, empty 9cm of the starch solution into each of the four test tubes. Place the tubes in a rack.
Using a 0-1cm syringe, add 1cm of the 1% amylase solution to one of the four test tubes and mix the contents.
Immediately start the stop-clock and after withdrawing up to 1cm of the mixture to the first test cavity in the spotting tile using the 0-1cm syringe.
Thirty seconds later, add 0.1cm of the mixture to the second cavity.
Repeat this progress every thirty seconds until a mid-brown colour is achieved and record the time taken.
Repeat steps using 2%, 3%, 4% of amylase concentration and record results.

The technique I used was very simple because of the limited amount of time and the lack of equipments available. I realised that if I had more time and better equipments I could of conducted a different experiment………

To ensure the investigation is fair I will:

Maintain the same volume of starch and amylase solution. This will make sure all the tests are identical which means the experiment will be fair. Therefore I do not need to change the temperature, ph, and the substrate concentration.
Use different syringes when filling the amylase and starch solution which will prevent contamination between the solutions.
Using a partner I will immediately start the stop-clock after the two solutions have been mixed. This will ensure the accuracy of the experiment, as the time will not be lost when the solutions are added.
I will also place two drops of the mixture after 30 seconds in each spotting tile using a pipette as this will ensure that equal volumes of solution is added.
With the assistance of my partner I will be informed when to place the two mixtures in the spotting tiles as if I was working alone I may lose valuable seconds and inaccurate results.

Safety

The most important part of the experiment is safety of yourself and others; this meant I needed to take serious measures.

First of all, the use of iodine is very dangerous as iodine is an irritant, so safety glasses must be worn at all times and care must be taken. An apron must be worn at all times for the protection of your skin and clothes.

Bags and stools must be kept out of the way or under a desk to prevent accidents such as falling down.

Results

Table 1 – The breakdown starch using 4 different concentrations of amylase solution

Table 2 – The time taken for starch to break down using different amylase solutions.

Graph – see graph paper.

Conclusion

From looking at the data obtained, my prediction was correct as an increase in concentration increased the rate of reaction. This can be seen in my results because the time taken to breakdown starch decreased as the enzyme concentration increased. This is due to the fact that more active sites were be available as more enzyme molecules are present, therefore more substrate molecules will be broken down. The rate of reaction will be slower if a lower concentration of amylase solution is used; this is due to the fact that there would be a competition for the substrate to react with the active sites. Also, my graph supported the my prediction that the rate of reaction was directly proportional to the enzyme concentration because more enzymes were present, therefore more active sites would be available for the substrate to slot into.

Evaluation

The experiment conducted was very successful although various variable were not controlled therefore the test lacked validity. I did not control the temperature that may have been a factor responsible for the increase in reaction. To improve this method, instead of using room temperature (room temperature varies), I could of used a thermometer to get one temperature in which the experiment could have been conducted. The shaking of the two solutions is not reliable as one solution may have been shaken more then another, which effects the rate of reaction because more molecules may have been colliding more in one test tube then another. I could also question the reliability of the results because only one set of results was obtained. I could improve this by replicating the experiment to obtain more consistent results.

Also, the measurements of the two solutions may have been inaccurate, as we did not have enough time to conduct the experiment.

The reading of time may not have been precise as it takes time for the solution to be taken into the syringe and dropped in the white spotting tile. I could have improved this by telling my partner to inform me a few seconds earlier to get ready rather then doing it on the spot.

A major problem is the judgements made on the colour. It was very difficult to distinguish between the colour of mid-brown. At once stage, my partner and me were disagreeing against the colour of the solution in the spotting tile.

Finally, to improve the experiment I could of used various techniques that may have been more suitable …………….