Investigating the Rate of Reaction of the Enzyme Amylase on starch

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Osman Khan        Lee 13        Page  of

Individual Investigation:

Investigating the Rate of Reaction of the Enzyme Amylase on starch  

By Osman Khan

Lee 13

Aim

        The aim of my investigation is to see what factors affect the rate of reaction of the enzyme amylase on starch. These factors will be temperature, effect of an inhibitor, the enzyme concentration and the substrate concentration.

Starch

        Starch is a mixture of amylose and amylopectin. Amylose is made up of many monosaccharides units of ά- glucose molecules, joined by alpha 1,4 glycosidic bonds. This builds up a long unbranched chain of glucose molecules, which then coils around itself into a helical structure- rather like a spring. Amylopectin is similar to amylose but also has alpha 1,6 glycosidic bonds, which makes the chains branched. The mixture of these two molecules is starch and is used as energy storage in plants.

 Left: Diagram showing the structure of amylopectin

  Right: Diagram showing the structure of amylose

The result of the enzyme amylase on starch is the conversion to maltose. This result can be detected by using potassium iodide. If no starch is present the colour of the solution will turn orange. In the presence of starch the potassium iodide turns a dark blue/black colour.

        As I said earlier the starch molecules curl up into structures like a spring. The hole, which runs down the middle of this spiral, is the same size and shape of the potassium iodide molecule, so the molecules can fit into the spiral. This is what gives the intense blue/black colour when potassium iodide is mixed with a solution of starch.

        Due to the colour change of the solution, the transmission of light through the solution will vary. Therefore, using a colorimeter, I will be able to detect how the transmission of light through the sample changes. This can then be interpreted on a calibration curve of known starch concentrations, enabling me to find out how much starch is left after a given time period. I will therefore be able to calculate the rate of the reaction by measuring how much starch is still remaining.

Apparatus and Procedure Justification

        In my investigation I have decided to use some types of apparatus over others as I feel it will make my experiments more accurate and reliable. These types of apparatus are mentioned below:

  • Colorimeter

                A colorimeter works on the basis that it detects light intensity passing through a solution. In my experiments I will be able to observe colour changes when I add the potassium iodide indicator. The solution will initially start dark blue/black, as there will be more starch at the beginning of the experiment. It will then turn orange as the starch disappears- due to it being converted to maltose. As a result, I can measure the change in colour by determining how the light passing through the solution changes. This procedure is much better than observing the intensity of colour by eye, as it is more accurate and gives a numerical value.

  • Water Bath

                I have decided to use a water bath as oppose to a Bunsen burner, as there is a smaller fluctuation of temperature. Using a Bunsen burner to maintain a temperature is very difficult and also inaccurate. However, a water bath can maintain the temperature to a much higher level of accuracy. This will ensure the experiment is fair throughout, in terms of the temperature of the reactants.

  • Accurate Measuring Scales

                In my experiment I am relying on the measuring scales when making up a solution of various concentrations. Therefore I must use a balance that is very accurate. The percentage error of any piece of apparatus is calculated by

        

        (Error / Reading) x 100

The way to reduce percentage error could be done by making a larger reading- i.e. instead of measuring out 1g, measure 10g. This would therefore give you a smaller percentage error. However, in my experiment, I have to add fairly small quantities of amylase/starch solid, to make up my solution. In theory I could add more solid and dilute it in more solution, but this would mean there is a lot of wastage of chemicals. Hence, to reduce the percentage error, I must use a piece of apparatus with a smaller error margin. Using a measuring scale, which measures to 2 decimal places would be adequate, but if a more accurate measuring scale could be used, this would make my experiment more accurate.

  • Rinsing apparatus

                When I make up a solution of amylase, I will ensure that everything I prepare the solution in is rinsed out. This will prevent any contamination of my solution. Also, by doing this all the starch or amylase powder will get transferred to the final solution, which will ensure that the concentration is accurate. This will therefore make my experiment fairer and also it will provide me with more accurate results.

  • Volumetric Flask

                When I am making up the standard amylase 5% and starch 1% solutions, I will make these up in a 250cm3 volumetric flask. This is because the volumetric flask is a very accurate way of making up a solution of any concentration. It has a very low error margin and this will help me to get accurate and reliable results.

  • Burette

                When I am carrying out the experiment into investigating the effects of concentration on the rate of reaction, I will add the distilled water using a burette. This is better than using a measuring cylinder, as once again the burette has a low percentage error, and can be read to a greater degree of accuracy.

 

Making up a solution of amylase/starch

  1. I will rinse out any apparatus that I am going to use. This will ensure that when the solution is made there is no contamination.
  2. I will weigh out the amount of amylase/starch solid that I require into a beaker using accurate measuring scales- i.e. to at least 2 decimal places of a gram. The amount of amylase and starch required for each variable is shown later.
  3. Looking at all the variables, I require 570cm3 of both amylase 4% and starch 1%. For this reason I can make these concentrations up using a 250cm3 volumetric flask.
  4. Using a glass rod I will then dissolve the solid in a fairly small volume of distilled water.
  5. I will then transfer the contents of the beaker to the volumetric flask, using a funnel to prevent any of the solution from spilling.
  6. Next I will rinse out the beaker using a small amount of water and also add this to the volumetric flask. This will ensure none of the solid is left behind in the beaker. I will also rinse out the funnel.
  7. Once this is done I will fill the volumetric flask up to the graduation mark, making sure that the bottom of the meniscus is just touching the line.
  8. Ensuring the lid is on, I will then invert the volumetric flask several times so that the contents thoroughly mix.
  9. As the volumetric flask is only 250cm3 and the total solution of required amylase 4% and starch 1% is 570 cm3, I will have to do this procedure again when I run out of the solution.
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When varying the concentration of either amylase or starch, I do not require such a large volume. Using a 250cm3 volumetric flask for just making up 30cm3 of a solution would mean a lot of solution is wasted. Therefore the solution will just be made in the beaker in which the solid is weighed out. However, to ensure that the total volume of water added to the solution is accurate, I will use a burette to add the water. This procedure is shown below.

        

  1. As above
  2. As above
  3. Using a burette I will add the required volume ...

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