For this investigation I am going to investigate the effect of the concentration of an enzyme, in this case amylase, on the time taken for the enzyme to fully breakdown the substrate, which in this case is starch.
PLANNING
For this investigation I am going to investigate the effect of the concentration of an enzyme, in this case amylase, on the time taken for the enzyme to fully breakdown the substrate, which in this case is starch. The reason why these two substances have to be used is because each enzyme is designed specifically to break down only one substrate as each enzyme is made of a protein that causes it to be a specific shape, in this case the enzyme amylase can only break down starch to dextrin. I am going to do this by planning an experiment, carrying out the experiment, recording any relevant results and plotting graphs from which I will be able, to gain a strong conclusion. I will finally evaluate the whole investigation.
Before the experiments start we will be given a bottle containing a 1% suspension of starch and another bottle containing a 1% solution of the enzyme amylase. From these bottles I will be able to take out as much of either solution as I wish. I cannot start the experiment until I know which factors I am going to change and which I am going to keep constant. Because the variable I am trying to test is the effect of the concentration of the enzyme, the only thing that I should be changing in each experiment is the concentration of the enzyme and nothing else. This will make all of the tests identical (apart from the enzymes concentration) which means the experiment should be accurate and fair. Because of this any differences in the results of my experiment I will be able to put down to the concentration of the enzyme. If I was to use two variables, and there were a difference in the results, e.g. I changed the temperature as well as the concentration of the enzyme and the reaction speeded up, I would not know which of the factors had done this and therefore could not reach a valid conclusion. Things that have to be kept constant throughout all of the experiments are:
Temperature
It is vital that the temperature remains the same in all the experiments, as this will have a great impact on the results if not. This is because if the temperature increases the amylase and starch molecules will begin to move faster due to the kinetic theory. (This is explained in the introduction section) Because of this, amylase molecules will bump into and come into contact with the starch molecules more often causing the starch to be taken up by the active sites of the amylase, broken down quicker and the product dextrin released. This will lead to an increase in rate of reaction. If however the temperature rises too much (above about 60 degrees) the ionic and hydrogen bonds holding the amylase together will break causing the active sites on the enzyme to become denatured, this will completely stop the enzymes working and therefore the breakdown of starch will stop thus causing the rate of reaction to stop.
pH Level
This is once again like temperature in the way that every enzyme has an optimum temperature the same as they have an optimum pH. Optimum means the "best" or in this case the best conditions for the enzyme to break down the starch the quickest. If the pH level is at an extreme (in this case strong alkali) the enzyme will be denatured and work at a slower rate or even stop. For these reasons the pH will remain the same throughout the experiments so as not to change the rate of reaction. In a more complicated experiment a pH buffer may be used to be certain the pH level would remain constant.
Concentration of substrate
If there are more enzymes (amylase molecules) than substrate (starch) molecules and then more substrate is added the rate of reaction will increase because there will now be more active sites available for the starch to be broken up in but if there is enough substrate to occupy all the active sites on the enzymes then the rate of reaction will not change and for this reason I will use the same volume and the same concentration of substrate for all of the experiments. This meaning all the concentrations of amylase will be working ...
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Concentration of substrate
If there are more enzymes (amylase molecules) than substrate (starch) molecules and then more substrate is added the rate of reaction will increase because there will now be more active sites available for the starch to be broken up in but if there is enough substrate to occupy all the active sites on the enzymes then the rate of reaction will not change and for this reason I will use the same volume and the same concentration of substrate for all of the experiments. This meaning all the concentrations of amylase will be working on the same amount of starch.
Inhibitors
Competitive inhibitors compete with the substrate for the same active sites on the enzyme this means the reaction will be slower as the starch will have less active sites to go into, where as non-competitive inhibitors compete for different sites but change the shape of the enzyme they attach to thus making the active site inactive and the starch molecules now cannot fit into the new shape of the active site. In both cases the rate of reaction will be slowed down. This should not be a factor in my investigation, as no inhibitors will be used in any way.
Volume of amylase and starch solution
This has to be the same in each experiment as differences would cause there to be a different number of either starch or enzyme molecules which would have the same effect as a different concentration of substrate. The thing that I will be changing in order to make the investigation work is the concentration of the enzyme solution. This is done so any change in the time taken for the amylase to break down the starch can be said to be because of the concentration, which reinforces the aim of the investigation. I will do this by diluting the 1% amylase solution to the desired concentrations. The concentrations I am hoping to use are 0.2%, 0.4%, 0.6%, 0.8% and 1%.
Volume of iodine
The volume of iodine solution I will be using is not going to be as precise as the other constant variables because I will not be able to use a measured pipette. Yet still, in order to approach some reliable points I will use the number of drops of iodine solution for each concentration, hence, I will use only one drop of iodine solution. I chose one because if I use 2 drops of iodine, this will risk keeping the volume of iodine constant because we will not be able to know if the second drop will be equivalent to the first drop: I have decided to use only one drop of iodine solution.
Information on my reaction
Amylase, like other enzymes, works as a catalyst, i.e. it is unchanged by the reaction, but makes the reaction easier by reducing the energy required for it to happen. Catalysts speed up the reaction. The theory behind the working is called the "lock and key" theory: the enzyme is shaped so that the products fit into them, react and are released (as discussed previously). Amylase digests starch by catalysing hydrolysis, which is splitting by the addition of a water molecule. Therefore starch plus water becomes maltose (which is equivalent to two joined glucose molecules).
There are two kinds of amylase enzymes. Alpha-amylase is found in saliva and is called ptyalin. This can carry on working in the stomach for several hours (and can digest up to 40% of starch under correct conditions of stomach acidity and food solidity). The other kind is called pancreatic amylase and is secreted in pancreatic juice, into the small intestine or ileum. Other enzymes then further digest the maltose to glucose and this is then absorbed through the wall of the small intestine by the body to be used as energy after being taken to the liver.
IMPLEMENTING
Apparatus and procedures to be used listed
% salivary amylase : I had to use salivary amylase because it was easy to acquire and could be easily kept fresh by placing it in a fridge when it is out of use.
% starch : starch was used because of the basic role of the constituent which is in this case the salivary amylase to initiate the digestion of starch to maltose. Additionally, to overcome an obstacle I had to dilute the starch, too in order to maintain the accuracy. For instance, we had 10 cm3 volume of starch but to dilute it down to 1%, I mixed 5.0cm3 starch and another 5.0 cm3 of water in to a test tube, therefore, we could add the percentage of the concentration of the starch up to 1.
5 x 10mm diameter test tubes : These will be used for diluting the solutions and for Clean test tubes mixing the amylase with the starch in the water bath. The test tubes have to be clean to prevent any unwanted contaminants getting into the experiment.
3 x 10ml syringe : Used for very accurate measuring of both the amylase, starch solution and water. This will be vital for getting the correct volume of the 3 substances into the test tubes.
Thermometer : The most accurate way of measuring the temperature of the water bath and therefore the temperature of the enzyme and substrate.
Stopwatch : To measure accurately the time it takes for each to 10th of a second concentration of amylase to break down the starch.
250cm3 : for the water bath because it is large enough to hold the water, 2 test tubes and the thermometer at the same time. It also conducts heat well.
Bunsen burner
Heat Proof mat, tripod Test tube rack : To hold the test tubes before and after the reaction has Tongues been carried out.
Iodine solution : To test for the starch as iodine turns from a reddish orange color to purple- black if starch is present (with dropper.)
Pipette : to take a tiny volume of the solution out of the test tube during the time when the enzyme will be breaking down the starch.
Making the amylase concentrations as the amylase solution that is given is already at 1% I will not be able to concentrate the solution any higher without difficulty for this reason I have decided to lower the concentration of the enzyme to my desired levels. To do this I will have to have, depending on the concentration needed a ratio of water - amylase solution. I want to make 10cm3 of all the above values. E.g. to make a 0.5% concentration of amylase I will need 5cm3 of distilled water and 5cm3 of amylase solution.
Method
Amylase Concentrations 1%
.00
0.80
0.67
0.50
0.25
The concentrations needed for each solution I will use are shown above. (TABLE) These will be made by using the syringes to collect the amylase and water. Different syringes are used for different substances, it is important that the concentrations are accurate. To make clear myself, I am now going to explain how I approached these readings: first, I used undiluted enzyme solution, secondly, I used half diluted enzyme solution(5.0 cm3 of amylase is added on to 5.0 cm3 of water-giving me a percentage of 0.50 of amylase concentration), thirdly, I took starch with a quarter diluted amylase concentration-giving me the percentage of 0.25 of amylase)then I took the starch with 3 quarters diluted amylase concentration-giving me 0.67%),finally starch with four fifths diluted amylase -giving me the percentage of 0/80).
They will then be put into a test tube, which will be slightly shaken to make the solution mixed equally. First the amylase solution has to be mixed to the needed concentrations. Then taking the test tube with the 0.2% amylase in it suck up 2.5cm3into the syringe. Make sure to hold the syringe at 180 degrees to the test tube so the measurement is accurate. This can then be put into a test tube. Using the same method with a separate syringe draw up 2.5cm of starch suspension, which can then be placed into a test tube.
Both the test tubes have to be placed in a water bath so that both the enzyme solution and the starch are at the same needed temperature of 40 degrees oC.
Heat the water in the beaker using a Bunsen burner until the temperature is at a constant 40 degrees. Keep the temperature at this for about one minute so the starch and amylase solutions have time to reach this temperature. (i.e. check with thermometer) Then being vitally careful not to spill any solution add the enzymes solution to the starch and slightly shake the test tube. Start the stopwatch as soon as the solutions are mixed. Now the solutions are mixed slight help may be needed to keep the temperature at 40 degrees while I will be checking the solution to see when the amylase has completely broken down the starch. I will do this by taking only one drop of the solution out of the test tube, using the pipette, and dripping it onto a spotting tile. I will then add a tiny drop of iodine to the starch solution using the provided dropper. If starch is still present the iodine will turn a blue/black color that shows me that the amylase has not completely broken up the starch. I will repeat this process every minute until eventually the iodine does not change color this will mean the amylase has broken the starch up fully and the stopwatch can be stopped. The results will need to be recorded in a suitable results table so I will be able to analyze them later.
Here is an overall view of my experiment to show the practical steps:
Precautions
Throughout the experiment, safety precautions should be taken to ensure that the experiment was conducted in a safe manner: safety goggles and a lab coat would be worn at all times. All parts of the experiment would be undertaken with care, to ensure that there was no spillage. Any spillages of iodine or starch would be washed thoroughly with soap and water. If any starch solution, iodine or amylase were accidentally ingested, medical attention would be sought immediately. Moreover, we stand up while using the Bunsen burner so we can move away quickly should the water bath fall. Any long hair tied back to keep out of way of flames
Hypothesis
As the enzyme concentration increases, it takes less time to break down.
Enzymes are very specific; they will only convert certain substrates into products. They do this by using either the "Lock and Key" theory or "Induction Fit" theory.
Both allow enzymes to work only on specific substrates and nothing else. The enzyme acts as a lock, and the substrate being the key. The area where the two molecules bind and where the reaction takes place is called the active site. The substrate is held to the enzyme by temporary bonds, which form between the substrate and some of the R groups of the enzyme's 'R' groups. The shape and size of the active site depends on the substrate molecule, so that both can fit together like a key that fits into a lock.
To act as a catalyst, the enzyme actually reduces the activation energy required to cause a reaction to happen. Giving the reactant molecules more kinetic energy can further increase this reaction - heating the substances causes the molecules to vibrate faster, thus increasing the chances of colliding with another.
In addition to increasing the likelihood of a collision, the chance of a 'successful' collision (one in which the molecules have enough activation energy to undergo the reaction) is also increased.
Activation energy is the initial energy required to trigger a reaction.
The graphs below show what activation energy is required for a reaction without and with enzymes.
Without
With
Reason for this hypothesis is that as the enzyme concentration increases, number of active sites increases also the rate of enzyme-substrate formation increases. The increase in the concentration leads the formation of more key and lock process. Because they get faster and quicker.
Predicted observations and graph
The graph I will be plotting will be of concentration of amylase against time taken for the starch to be broken down. I believe that the graph will show that as the enzyme concentration increases the time taken decreases.
I predict that the rate of reaction will increase as the temperature increases (having a positive correlation) until the reaction reaches an optimum temperature. Above this optimum temperature, the rate of reaction will fall to zero very quickly as the enzyme denatures.
The breakdown of starch will be quicker when the temperature is increased until it exceeds 40° C. Then the amylase will no longer catalyze the breakdown of starch.
Above, I indicated an example Exponential Decay Curve so that after every x minutes, the starch concentration would half and would therefore never be totally broken down. It was predicted that, if a graph were drawn to show starch concentration against time, it would be an exponential decay curve. The exponential behavior was predicted because the reaction is not an equilibrium reaction: as the starch concentration decreases the enzyme finds it increasingly difficult to find enough substrate to act on. Although using iodine to test for starch would prevent a graph from being drawn in this way, the trend would still be evident.
Furthermore, I have to conclude from the graph I will draw in the following page, that 1.00% concentration is the maximum in other words, the highest it can reach.
ANALYZING EVIDENCE
Concentration of amylase/
%
Time taken to digest starch
s
.00
0.00
0.80
.00
0.67
.50
0.50
3.00
0.25
4.00
Here are the readings of my data. Now, I should try plotting a scatter graph using these readings. Therefore, analyzing the hypothesis will be much more clear and precise to see according to the anomalous results omitted.
