Investigating The Effect of Substrate Concentration or Temperature On The Rate of Decomposition of Hydrogen Peroxide By Catalase In Immobilised Yeast
M1-Investigating The Effect of Substrate Concentration or Temperature On The Rate of Decomposition of Hydrogen Peroxide By Catalase In Immobilised Yeast
Problem:
Hydrogen peroxide is a waste product of the metabolism (chemical reactions of living cells). It is a powerful oxidising agent, which would damage living cells unless it was destroyed. Hydrogen peroxide will eventually decompose into un-harmful substances such as water and oxygen however in this slow process.
2H O 2H O + O
However the human body has a process which speeds up the breakdown of hydrogen peroxide. The human body produces an enzyme to speed up the breakdown of hydrogen peroxide; this enzyme is called catalase.
Hypothesis:
In my investigation I aim to explore one of the factors that would affect the catalysis of an enzyme; my research from textbooks, CD-ROMs (Encarta) and the Internet (www.britannica.com) tells me that factors such as; Temperature, pH, Substrate Concentration, Inhibition, Enzymes Cofactors and Enzyme concentration would greatly vary the rate of enzyme catalysis. However from this research I have decided substrate concentration (hydrogen peroxide) is the factor which would able my experiment to yield good conclusions. For this reason I have decided to look at how a change in temperature would affect the rate of a reactions.
Safety
As Hydrogen peroxide is a highly corrosive substance it is very necessary to wear protective eyewear and a lab coat and try to be extra careful when measuring out this substance
The yeast and the hydrogen peroxide are harmful to the body so I think it would be wise to wash my hands after I have completed my experiment.
As ever I will abide by all the lab safety rules
Fair Test
In my preparation of the alginate beads it is important to make sure that I pick a uniform size for the alginate ball as if they are massively different in size it means that it will take longer for the oxygen to lift the bead and also there would be more surface area for the enzyme decomposition to take place therefore speeding up the reaction.
In all my measuring it is important that I drop the alginate bead in from as close as I can get to the surface of the hydrogen peroxide concentration and that I do not vary this distance in any of my measurements.
The volume of my substrate should be kept as accurate as possible so I will use measuring a syringe and measuring cylinders
The rate of enzyme catalysis can be greatly affected by temperature so it is important that I work in one place throughout my investigation (i.e. not near a window/radiator)
In the preparation of my alginate beads (immobilised enzyme) I think that it is important to have an even spread of the enzyme throughout the bead, so I will mix it vigorously.
Scientific Knowledge
Enzymes exist in all living things. They are composed of polymers of amino acids and are produced in living cells. Each cell contains several hundred enzymes, which catalyse a vast number of chemical reactions. Enzymes are known as biological catalysts as they significantly increase the rate at which reactions occur within living organisms, without being used-up or affecting the reaction in any other way. Enzymes save the need for an increase in temperature in order to speed up reactions within living things. Minute quantities of an enzyme can accomplish at low temperatures what would require ...
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Scientific Knowledge
Enzymes exist in all living things. They are composed of polymers of amino acids and are produced in living cells. Each cell contains several hundred enzymes, which catalyse a vast number of chemical reactions. Enzymes are known as biological catalysts as they significantly increase the rate at which reactions occur within living organisms, without being used-up or affecting the reaction in any other way. Enzymes save the need for an increase in temperature in order to speed up reactions within living things. Minute quantities of an enzyme can accomplish at low temperatures what would require violent reactants and high temperatures by ordinary chemical means.
Enzymes are globular proteins and their molecules have a very precise three-dimensional shape, which forms a one specific active site on the enzyme. Each enzyme can only convert one kind of substrate molecule into one kind of product molecule. In this enzymes globular structure, one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site, or the location on the enzyme where the substrate binds and the reactions takes place. This specific ness enables enzymes not to participate in the wrong reactions. It is exactly the right size and shape for enzyme's substrate to fit into (in the case of catalase this is Hydrogen peroxide). When a substrate molecule slots into the active site, the enzyme 'tweaks' the substrate molecules, pulling it out of shape and splitting it into product molecules. In this way the enzyme catalase catalyses the anabolic/catabolic reaction of hydrogen peroxide. This is called an enzyme-substrate complex.
I know that the reaction rate is the speed at which reaction proceeds toward equilibrium. Then energy barrier between reactions and products governs the reaction rate, In general energy must be added to this reaction to overcome the energy barrier-this energy is called the activation energy. Enzymes accelerate the rates of reactions by lowering the activation energy. If there are sufficient enzyme molecules, an increase in the substrate concentration, will produce an increase in the rate of reaction
Prediction:
I predict that have there are sufficient enzyme molecules, an increase in the substrate concentration, will produce an increase in the rate of reaction. When the substrate concentration has been completely decomposed into hydrogen and water I predict that the rate of reaction will slow down considerably.
I predict that as the substrate concentration increases it will reach a saturation point where the enzyme is literally being smothered by the amount of hydrogen peroxide molecules and cannot increase the rate of reaction any more. Also because of this I think that the rate of reaction will level off (as in the graph above).
Plan:
In order to observe the effect of temperature on catalase I will be recording the rate at which the oxygen is produced in the decomposition of hydrogen peroxide. I have decided that my independent variable will be varying concentrations of hydrogen peroxide, the concentrations I have chosen will be- 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. I have decided to be able to measure the rate of enzyme catalysis I will measure this by timing how long it takes once the Alginate beads touch the surface of the hydrogen peroxide concentration and how long it takes to rise and float in my varying hydrogen peroxide (substrate) concentrations.
Step 1: Prepare the alginate beads by mixing a solution of 3cm of yeast, 3cm of sodium alginate and 3cm of air. The yeast in this solution is the source of the catalase. I have decided to use the alginate as my source for the enzyme-in this way the enzyme is immobilised. I have decided to test these alginate balls with 11 varying concentrations of hydrogen peroxide and I will also test these 11 concentrations 5 times and take an average. Because of this I will make around 40 alginate balls providing that all these balls are evenly produced. In this stage I will wear goggles and if I spill some of the hydrogen peroxide concentration on my hands I will immediately wash them because hydrogen peroxide is highly corrosive.
Step 2: Now I will test these alginate beads with each varying concentration of hydrogen peroxide. As an example, having picked an evenly prepared alginate ball I will drop it into the test tube containing a hydrogen peroxide concentration. With a stopwatch I will time from when I place the alginate bead into the concentration and time that it takes until the bead rises to float on the top of the hydrogen peroxide concentration. When I am timing the reaction I will have to be very responsive to keep my investigation accurate. Again in this stage I will have to be careful with the highly corrosive hydrogen peroxide concentrations.
Step 3: After having measured the rate of enzyme catalysis in response to the rate of the oxygen being given off, I will put these figures into my table. After having conducted this investigation I think that I would be in a good position to analyse my results and yield conclusions.
Fair Test:
Temperature: To control this variable I will try to my best to keep the temperature on all my enzyme and substrate molecules kept constant so that the results are kept constant. I will expect the room temperature will be around 18 degrees and I assume that that it will hardly change throughout the experiment.
pH: Any change in Ph affects the ionic and hydrogen bonding in an enzyme and so alters its shape. Each enzyme has an optimum shape at which the enzyme best fits the substrate. Variation either of neutral pH results in denaturation of the enzyme and a slower rate of reaction. Hopefully nothing in my investigation will affect the Ph of my enzyme-substrate molecules therefore keeping my results accurate.
Substrate Concentration: When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. I must make sure that the volume of each substrate concentration should me measured precisely with a measuring cylinder.
Enzyme Concentration: Provided there is an excess substrate, an increase in enzyme concentration will lead to a corresponding increase in the rate of enzyme catalysis. In this investigation the enzyme concentration will be kept constant provided that my preparation of my immobilised enzyme (alginate beads). This will be kept accurate because in the preparation of the alginate beads the yeast (source of catalase) will be well mixed.
Pressure: The introduction of pressure will provide less space for the enzyme-substrate molecules therefore meaning the collision theory will be doubled. Because of this the enzyme catalysis will be increased. Because I am going to be performing my investigation in the open air of the science lab I will have no means to control this variable. However pressure will not change greatly enough in my lab to change my results
Apparatus:
Step 1:
Equipment To Prepare My Immobilised Enzyme (Alginate Beads):
ml Syringe
3cm Yeast (catalase)
3cm Sodium Algenate
3cm Air
Sieve
Step 2:
Equipment To Measure Rate of Reaction (Rate of Catalysis)-Measure Oxygen By Product:
Stopwatch
1 Test Tubes
Varying concentrations of Hydrogen Peroxide- (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%
80%, 90%, 100%)
Distilled Water
Temperature
55 Alginate Beads
Test Tube Rack
Forceps
Analysis
From my table of results and from my graph of reaction rate (1/time) versus substrate concentration it is clear that as the concentration of the hydrogen peroxide increases, the time taken for the bead to reach the surface lessens. This means that with the increasing hydrogen peroxide concentration increases the rate of enzyme catalysis meaning overall that the rate of reaction increases.
It is clear that with the positive correlation that I have acquired with the substrate concentration versus rate of reaction my prediction that if there is sufficient enzyme molecules, an increase in the substrate concentration, will produce an increase in the rate of reaction
However from the graph above it clearly shows that the rate of reaction did not level off at the end proving that I hadn't reached a saturation point. However I do think that maybe the saturation point might have been reached if I had more of the substrate (catalase).
Evaluation
In the methods that I was conducting I feel that the most inaccurate method was the preparation of my immobilised enzyme (alginate bead). I feel that I measured out the sodium alginate, air and yeast very accurately with a syringe and a measuring cylinder. However I feel that in this preparation there may not have been an even mix of the catalase (yeast) in the alginate solution: ultimately meaning that the catalase concentration in each alginate ball would have been very varied-changing my results somewhat. I did mix this solution vigorously and my preparation was very accurate and the catalase was to an extent, evenly distributed.
However I feel the mixing of the solution is done in a crude way and my results would have been more accurate because of this. I also feel that if there was a way in order to accumulate 55 alginate balls of the same uniform size my results would have been much more accurate. Therefore in the reaction of enzyme decomposition each ball would have a uniform surface area for the reaction. Also the size of the bead wouldn't determine the time the oxygen takes to lift the bead to the surface.
In my experiment I used the same hydrogen peroxide concentrations for each of the 5 tests simply because there weren't enough test tubes and concentrations for the whole of the class to use. So in each test it could of meant that there would have been less and less hydrogen peroxide molecules in the concentration for the enzyme to react with ultimately meaning that the rate of reaction would have become slower and slower in each test. If I were to extend this investigation I would definitely use a different hydrogen peroxide concentration each time.
I think that because I have measured the enzyme decomposition five times and have taken an average from this I feel my results are very reliable, the fact that I had repeated it 5 times means that any mistakes I made I could have looked at, rendered the mistake and repeated the test. Within the limits of how accurate I could keep my experiment I was very rigorous and because of this I feel I have managed to yield evidence to support the prediction that- if there are sufficient enzyme molecules, an increase in the substrate concentration, will produce an increase in the rate of reaction
To improve my results I could start by testing the enzyme decomposition with a wider range of hydrogen peroxide concentrations as this would better my timings for the average rate of reactions.
