Determination of the effect of enzyme concentration on catalysis using starch an amylase.
INTRODUCTION
Enzymes are said to be catalytic proteins which increases the rate of a chemical reaction without being altered in the process of that reaction. [1] A substrate is a substance which an enzyme acts upon. No bond is formed between the enzyme and the substrate in the reaction thus the enzyme goes back to its original shape and can be used again.[2]An enzyme binds to a substrate via the active site thus forming an enzyme – substrate complex They are very specific in their reaction and also to the substrate they are binding with. Enzymes function correctly when the shape of the substrate matches the enzyme’s active site and their functioning is dependent upon its three dimensional structure. They undergo catalysis by lowering the activation energy so that more molecules will be activated thus having the reaction occurring more easily [1] [2]
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In this experiment amylase is use to break down the starch molecules. Starch is the substrate used and amylase is the enzyme. There is a change when amylase reacts with starch. There is a release of a disaccharide – maltose. As time increases there will less abundance of starch and more of the sugar present. So when this is added to iodine the blue/black colour will decrease to a light yellow shade.[4]
The concentration of the enzyme is important in chemical reaction as it is needed to react with the substrate. Often a small amount of enzyme can consume a large amount of substrate. But as enzyme concentration increases so is the availability of active sites thus these will convert substrate molecules into products. What this is basically saying is that if the enzyme concentration is to be increased there needs to be an excess of substrate present which in other words means that the reaction must be independent of the concentration of substrate.[3]
Apart from the concentration of substrate and enzyme there are other factors which can also influence the enzyme to function to its optimum capacity. These include temperature, pH, and inhibitors. Higher temperature would allow for more collisions to occur therefore allow substrate to bind to the enzyme’s active site more frequent. Since enzymes work at a certain temperature range activity would decline once this range would have been exceeded and the enzyme is denatured. Each enzyme has its own optimum where it functions best. Pepsin, an enzyme found in our stomach, works best in acidic conditions. Some enzymes becomes denatured thus deactivated when pH goes up down.
I predict that the rate of the reaction will increase as the concentration increases and vice versa. The reaction will occur fast once the enzyme is added but it will slow down upon descending to the last test. I also believed that only a few of the test tube will produce a blue/black colour since the starch present in the solution will be hydrolyzed.
Apparatus/Materials
Water
Buffer solution ( pH 6.8)
1% starch solution
1% amylase solution (Saliva)
Dropper
3 beakers
3 10 ml measuring cylinders
12 test tubes
Test tube rack
Timer
Method:
Four test tubes were labeled A – D
2 ml of water was measured and placed in test tube A. 2 ml of amylase (saliva) was measured and placed in the same test tube.
Again 2 ml of water was measured and placed in a second test tube, test tube B, and to this 2 ml of the solution in test tube A was added.
Another 2ml of water was added to a third test tube, test tube C , and to this , 2ml of the solution from test tube B was added.
A further 2ml of water was added to test tube D, and to this 2 ml of solution from test tube C was added. Two milliliters of solution from test tube D was discarded so that all will have equal amounts of solution.
Forty drops of buffer solution was added to test tube A .
Eight (8) test tubes were collected and placed in a test tube rack. Two drops of iodine solution was placed into each using a dropper.
To tube A 0.5 ml 1% starch solution was added.
One drop of solution from tube A was immediately transferred to test tube #1 containing iodine solution. The dropper was properly rinsed.
After 1 minute, one drop of solution from tube A was added using the dropper to the second tube containing iodine. The dropper was rinsed thoroughly. This was done for all the other test tubes that remained.
The contents in all eight iodine test tubes were discarded. The tubes were thoroughly rinsed and dried for use in the next round of tests.
Steps 6 – 11 was repeated for test tubes B,C,and D.
RESULTS
Test Tube
Test Tube with Iodine
Observations
A
B
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Dark brown solution with small amounts of blue/black grains. These were apparent 17 seconds after adding solution A
Dark brown grainy solution.
Orange brown solution with particles which were also orange -brown
Light orange – brown solution. No grainy particles present
Lighter orange – brown solution
Yellow – brown solution
Yellow brown solution. This was lighter than tube No. 6
Light yellow brown solution. This was exceptionally lighter than the others.
Blue- black with coarse particles. Small traces ( 320 seconds)
Orange brown solution
Light orange brown solution with grains present
Orange brown solution with tiny grains present
Orange brown solution
Orange brown solution
Light orange brown solution
Light orange brown solution
C
D
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Dark brown with small traces of black particles (fewer than with tube B)
(455 seconds)
Orange brown solution
Orange brown solution
Orange brown
Dark orange brown
Dark orange brown
Very dark brown solution with a few grainy particles
Very dark brown with lots of grainy particles
Dark brown solution with very small traces of black grains ( 560 seconds)
Dark orange brown, no grainy particles present
Dark orange brown solution
Orange brown solution
Orange brown solution
Yellow/ orange- brown solution
Yellow brown solution
Light yellow brown solution
The graph shows how the concentration of the enzyme affects the overall rate of the reaction. A higher concentration of the enzyme will produce a faster occurring reaction than a lower concentration. From the graph as time proceeds the reaction rate drops significantly.
DISCUSSION:
This lab exercise demonstrated the ability of an enzyme to hydrolyze the substrate molecule. The enzyme used was amylase and the substrate was starch. The starch is what the amylase actually acts upon to give the end products i.e amylase breaks down starch.
Substrate ENZYME Products
Enzyme concentration and substrate concentration play a vital role in enzymatic activity.
The more enzymes available, the quicker the reaction will occur until the substrate is all used up
More substrates will also mean quicker activity, until the enzyme is fully saturated so that it
cannot continue increasing its activity.[1]
Based on the results obtained from tube A, a blue/black colouration was noted. This indicated that there was significant amount of starch present. Iodine is an indicator for the presence of starch. This same colour was noted for tubes B- D but the traces of blue /black colour decreased from tube A -D. As the tests proceeded to the last tube, the colour of the solution for each set changed from a dark brown solution to light yellow and in some cases to a light orange brown solution.
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A reasonable explanation for this is that there are fewer enzymes present as you move from tube A-D thus the starch will not be broken down. When there is an insufficient amount of enzyme present the reaction will not progress as quick as it would because the active sites present are occupied. If the concentration or amount of enzymes is increased then this would make provision for an increase in reaction rate. Reaction rate would increase due to the fact that there will be more active sites that are unoccupied. However, if there is an excess of enzyme molecule, the rate would not increase if more is added but it would reach at a point where it would level off.[2]
Another reasoning behind the colour change in that after the amylase reacted with the starch there will be a discharge of maltose which is a disaccharide. Less starch will be present as time proceeds and more maltose will be present. In addition less starch will be available to react with iodine thus the blue/black colour will decrease.
The predictions made were moderately correct since a lower concentration of enzyme produced a reaction which was slow and one that had less products being formed.
Various factors could have affected the results of the lab which may have given some amount of inaccuracy. These include temperature and pH. The enzyme perhaps would have functioned better in a certain temperature range instead of normal room temperature.
CONCLUSION
Based on the results obtained from the experiment it can be concluded that the concentration of enzymes influences the rate of a chemical reaction. If enzyme concentration is decreased then the reaction rate will also decrease. If there is sufficient enzyme to bind with substrate then the reaction will proceed fast and if there are insufficient enzymes present then the reaction will slow down
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