How does varying bacterial amylase effect inhibition by copper?

University Degree Biological Sciences

INVESTIGATION

Amylases are enzymes, which hydrolyse starch into maltose. There are two main types of amylase: -

Alpha amylase, which degrade starch molecules into fragments 10 glucose residues long;
And there are Beta amylases, which break down into maltose made of two glucose molecules.

Both are working by hydrolysis adding one molecule of water across the glycosidic link.

Hypothesis

I think that the higher the concentration of bacterial amylase, the faster the rate of reaction.

This is because the higher the concentration the faster the rate of reaction. Increased concentration results in an increased possibility that collisions between molecules with the required activation energy will occur.

Copper is non-competitive inhibitor. In non-competitive inhibition, the inhibitor may form a complex with the enzyme itself, with the enzyme/substrate complex or with the prosthetic group. The inhibitor is not competing for the active site but joins to the enzyme molecules elsewhere.

Bacteria use amylase to feed and therefore provide energy to the cell. Bacteria are prokaryotes. Prokaryotes are made up of prokaryote calls. They do not have a membrane-bound nucleus. The genetic material is in a single strand coiled in the centre to form a nucleiod. They are also single celled organisms. We cannot live without bacteria, yet bacteria causes may diseases, which kill many people, for example TB, typhoid, cholera, and many more.

Bacteria can be found in a wide range of environments, for example, water, air, soils and sediments.

Bacteria are also found in a wide range of temperatures from 0-90 °C.

The bacteria have adapted to the environments. Therefore I think that bacterial amylase have a better chance at withstanding changes in temperature and pH and therefore will be more resistant to denaturation.

The rate of reaction will increase as the temperature increases. This is because the molecules have more kinetic energy resulting in more successful collisions. However, after the initial increase in the reaction rate, as temperature continues to increase above 45 °C, The enzymes will become denatured. This is because they consist of protein and have specific, complex 3D structures. As temperature increases the enzymes become progressively inactive so they can no longer catalyse the reaction. This process of denaturation is the irreversible destruction of the tertiary structure of the enzyme protein, changing its complex 3D structure (i.e. shape) and eventually destroying its active sites and therefore cannot produce an enzyme/ substrate complex.

The active site is a specific region on the protein molecule where the substrate binds to the enzyme and so their shape is vital to the whole process. When the shape is altered, the substrate can no longer fit into the active site to produce an enzyme/substrate complex. So I need to find the optimum temperature for the reaction.

Plan

I plan to use different concentrations of bacterial amylase and add a fixed amount of starch and copper sulphate and time how long the experiment takes.

Starch can be detected by iodine solution. The reddish-brown colour changes into a blue-black when the starch is present. The blue-black colour is my start point and this should turn colourless when the starch has changed into maltose.

Maltose is a sugar and can be detected by Benedict’s solution. When a reducing sugar is present then Benedict’s solution changes from sky blue to a brick red precipitate.

Key variables

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Plan

I plan to use different concentrations of bacterial amylase and add a fixed amount of starch and copper sulphate and time how long the experiment takes.

Maltose is a sugar and can be detected by Benedict’s solution. When a reducing sugar is present then Benedict’s solution changes from sky blue to a brick red precipitate.

Key variables

Starch concentrations and volumes will be kept the for all the experiments;
The same amount and concentrations of iodine will be used for all the experiments;
The same concentrations and amount of copper sulphate solution will be used for all the experiments;
The same volume of bacterial amylase will be used for all the experiments;
Buffer solution will be used to eliminate any pH changes.

Risk assessment

Amylase

These are enzymes and have biological activity and should be treated with care. Must be clearly labelled. Avoid spillage on skin. If this occurs, wash as soon as possible and wear eye protection.

Copper sulphate solution

Toxic and irritant therefore eye protection must be worn. Avoid spillage on skin. If this occurs wash as soon as possible with water. Must be clearly labelled.

Iodine solution

Toxic and irritant therefore eye protection must be worn. Avoid spillage on skin. If this occurs wash as soon as possible with water. Must be clearly labelled.

Starch solution

Low risk but keep in a labelled container.

Preliminary experiments

However I need to do a preliminary experiment to find out the concentrations, temperature and the pH of the experiment to obtain the best results. From previous experiments and research from books I can see that bacteria can survive at a range of temperatures and pHs. So for the preliminary experiment I will use room temperature and pH 7.

I want to find the concentration of copper sulphate solution to use for the main experiment and also what volumes of starch solution to use.

I got 6 test tubes and add 2ml of 2% starch solution to each test-tube. Then I added 2 drops of 1% iodine solution to each test-tube. This is the test for the starch. The starch changed into a blue-black colour. Next I added 1% copper sulphate solution to the 1st of the test tubes and labelled the test-tube 1%. Next I added 2% copper sulphate solution to the 2nd of the test tubes and labelled the test-tube 2%. Next I added 3% copper sulphate solution to the 3rd of the test tubes and labelled the test-tube 3%. Next I added 4% copper sulphate solution to the 4th of the test tubes and labelled the test-tube 4%. Next I added 5% copper sulphate solution to the 5th of the test tubes and labelled the test-tube 5%.

But for the last test-tube I did not add copper sulphate solution as this is the control.

Then I added 2ml of 0.25% bacterial amylase recording the time at which the bacterial amylase was added. To do the timing I used stopwatch that could measure to the nearest 0.01 of a second. I also added the pH 7 buffer solution.

Results tables

The results from this test could not be obtained, as the iodine solution was too concentrated. Therefore I am doing another preliminary test with lower concentration of iodine solution. I will use 2ml of 0.25% bacterial amylase, 2ml of 2% starch and 0.1% iodine. I will not add copper sulphate solution and the temperature will be at room temperature.

This shows that the experiment works but the experiment takes too long so I will repeat the experiment at different temperatures. The temperature range I will use will be from room temperature, 20 to 80°C. To do this I will need to set up water baths at different temperature.

This shows the optimum temperature for the reaction. The optimum temperature is in between 40 and 50°C so I will use 45°C. Therefore I will do the preliminary experiment I planned to do to find out the concentration of copper sulphate solution to use.

This shows the results for the preliminary experiment to find out the concentration of copper sulphate solution to use. From the results you can see 1% copper sulphate solution takes the shortest amount of time. Due to time limitations I will use 1% copper sulphate solution. I will also use 1% copper sulphate solution so I can repeat the main experiment to get accurate results.

Method

First I got six clean test tubes and labelled the test tubes 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and control. I measured out 12ml of 2% starch solution using micropipette and added 2ml to each of the six test tubes. Then I added 3 drops of 0.1% iodine solution using a small pipette to each of the test tubes. I made sure that all the solutions were the same colour using a colorimeter. I then measured out 10ml of 1% copper sulphate solution and added 2ml to each test tube apart from the test tube labelled control. Then I set up the water bath at 45°C. I then measured out 6ml of pH7 buffer solution and added 1ml to each of the test tube.

I then measured out 2ml of 0.1% bacterial amylase using a syringe. I also measured out 2ml of 0.2%, 0.3%, 0.4%and 4ml of 0.5% of bacterial amylase.

Once the temperature of the water bath had reached 45°C I put the test tubes into the water baths and added 2ml of 0.1% bacterial amylase to the test tube labelled 0.1% and started the stopwatch and recorded the time. I then added 2ml of 0.2% bacterial amylase to the test tube labelled 0.2% and recorded the time. I then added 2ml of 0.3% bacterial amylase to the test tube labelled 0.3% and recorded the time. I then added 2ml of 0.4% bacterial amylase to the test tube labelled 0.4% and recorded the time. I then added 2ml of 0.5% bacterial amylase to the test tube labelled 0.5% and recorded the time. Then finally I added 2ml of 0.5% bacterial amylase to the control experiment and also recorded the time.

I then waited until the blue/black colour had become colourless and recorded the finishing times. The sixth test tube was my control experiment to see if the bacterial amylase worked. In the control experiment no copper sulphate solution was added.

Once all the test tubes had become colourless I added Benedict’s solution and heated the solutions. I did this to see if maltose was present.

I repeated the exact experiment twice more to check for odd results.

Results table 1

Results table 2

Results table 3

CONCLUSION

Main Trends And Patterns

From the results, I can see that the higher concentration of bacterial amylase, the faster the rate of reaction. The 0.5 % concentration of bacterial amylase was quickest out of the five test tubes containing copper sulphate solution. The average time taken for the 0.5 % bacterial amylase was 324 seconds. The next quickest reaction was 0.4% bacterial amylase, with an average time of 421 seconds. The next quickest reaction was 0.3% bacterial amylase, with an average time of 530 seconds. The next quickest reaction was 0.2% bacterial amylase, with an average time of 648 seconds. Finally, the slowest reaction, which was 0.1% bacterial amylase, which took an average time of 1043 seconds.

Explanation Of Results

Enzymes are globular proteins, which are consisting of coiled polypeptide chains. The enzyme has an active site which the substrate fits into and forms an enzyme substrate complex. The enzymes have a specific 3D shape, which is maintained mainly by disulphate bridges and hydrogen bonds.

I have noticed that the rate of reaction increases as the concentration of bacterial amylase increases. This is due to increased number of molecules with the required activation energy, resulting in increased productive collisions between the molecules and substrate.

However, in this case a non-competitive inhibitor was introduced. The inhibitor slowed down the reaction by binding with the enzyme. This changes the shape of the enzyme and as a result, reduces the rate of the reaction.

As the concentration is increased, there are more enzymes in the solution than there are inhibitors. This means that more of enzymes are involved in the reaction, therefore the rate of reaction increases. This indicates that my hypothesis ‘I think that the higher the concentration of bacterial amylase, the faster the rate of reaction.’ was correct.

EVALUATION

The main problem of my experiment was to determine the end point of the experiment was. The colour change from the blue/black to colourless was difficult to determine. Attaining the correct concentration of bacterial amylase was the difficulty I encountered whilst conducting the experiment. In order to attain the correct concentration, I had to dilute the bacteria amylase using distilled water.

Overall, I believe that the experiment was accurate and that I acquired accurate results. I repeated the experiment three times in order to achieve accurate results and also rule out the possibility of ‘fluke’ or chance results. The experiment produced no anomalies.

The results provided evidence that supported my hypothesis. I was able to accept my hypothesis to be correct and conclude that the rate of reaction increases with increasing concentration of bacterial amylase.

To broaden my investigation for example, I could: