For my first experiment I will put in 10 cm? of starch solution (the starch concentration in the solution is 5% and I will not dilute this) and 1cm? of diastase (the concentration of diastase in the solution is 2% and this will not be diluted either) into separate test tubes and put the test tubes into an ice bath to bring the temperature down to about 2?c and when both solutions are at 2?c add them together and keeping them at throughout the experiment at 2?C I will keep taking a sample of the solution every 15 seconds to test if the enzyme has worked. I will test for starch by dropping 2 drops of the solution onto a spotting tile with iodine solution and will compare it directly to a pure starch solution test to see if there is even a slight colour change. I will stop my experiment after 20 minutes (I only stop as I would run out of time otherwise) and I will indicate in my results table that the enzyme has not worked in the 20 minutes that I tested it for.
I will repeat this experiment exactly the same as the first one but (without the ice bath for higher temperatures) having the solutions at different temperatures to find out what the optimum temperature/range is for the enzyme.
When I am doing the higher temperatures (100?c) I will heat a glass beaker full of water to the temperature I want then I will heat the enzyme and starch in their separate test tubes to the correct temperature then add them and keep the temperature the same. The reason I will heat the test tubes up in the water bath rather than directly in the flame is so the temperature is easily contained. If it was just in the test tube the enzyme could have boiled at 100?c the bottom and yet still be at 40?c at the top (and this would therefore be an inaccurate result)!
Results table key
No change = N
First sign of a change = 1
Light Brown coloured = 2
Yellow coloured = 3
Results table
Analysis
These are my results from the experiments I did. I think the optimum temperature for this enzyme to work at is 65?C. I think the enzyme was denatured at a much higher temperature of just after 75?C as the enzyme had worked but much slower than at previous temperatures there fore I think the enzyme would have been denatured at 80?C.
My experiments are limited to there accuracy as it is only of my opinion that there is a colour change and could be varied easily from person to person that does this experiment, I have however repeated my experiment to get the results as accurate as possible.
With the information I have got from my experiments I will say that the optimum temperature is 60?c. My prediction stated that I was expecting this to happen: at low temperatures very little or nothing would happen and at 1?c nothing did happen, at 25?c the reaction had speeded up a bit and had worked at around 15 minutes, at 40?c it was even quicker working at times around 11.45 minutes, 50?c (working around 9.40 minutes) and at 60?C it only took about 6.15 and so this was the highest temperature I did before the enzyme started to denature. At 75?c I think that the enzyme must have started denaturing as the reaction had decreased in activity quite rapidly.
My research states that most animal enzymes will be denatured by 45?c my experiment has shown it to be denatured above 70?C, and so as this enzyme has shown its activity rate to have peaked at 60?C so this enzyme (diastase) must be a synthetic one.
Evaluation
The optimum temp was 60?C. I have repeated all my experiments twice so they are a lot more likely to be accurate than if it was just done once, as all it would have only taken one wrong result to mess up my entire investigation. Mistakes will not be as likely because of the repeats. If I found an anomalous I would have repeated that experiment till I was sure that I had got it right. If I were going to do this experiment again with more accuracy if I could I would use a computer to tell if there is a colour change as that would be far more accurate than me deciding.
Procedure - I will label 5 test tubes at different temperatures as stated above leave one at room temp as control
I will place each test tube in appropriate water bath for 5mins exactly. Remove and cool rapidly to room temperature.
Requirements
5 test tubes labelled.
Test tube rack
Thermometer
Pipette (5cm)
Stop timer
White tile
6 glass rods.
Water baths maintained at 25, 40, 60, &100
25cm of 0.5% of Diastase solution. Starch solution as substrate 25cm of 0.5% dilute iodine.
Enzymes are proteins that control vital biological processes. They often act as biological catalysts. They are made in cells. A catalyst is a chemical substance which speeds up a reaction but does not get used up during it. One enzyme molecule may be used several times over.
A rise in temperature increases the rate of most chemical reactions and a fall in temperature will slow them down. In many cases a rise in 10? C will double the rate of reaction in acell. This is particle theory. I am going to investigate the temperature at which reactions occur. An increase in temperature will result in a n increase in kinetic energy. Since the kinetic energy increases, the velocity of particles will also increase. Since the speed of particles increases, they should collide more often and therefore the speed of reaction increases. The particles will also have more energy thereby speeding up the reaction even more. It has been suggested that for every 10degree rise in temperature, the speed of the reaction will double till optimum.
At low temperatures particles of reacting substances do not have much energy. However, when the substances are heated, the particles take in energy. This causes them to move faster and collide more often. The collisions have more energy, so more of them are successful. Therefore the rate of reaction increases. The more successful the collisions are the faster the reaction. The same can be said for reactions controlled by enzymes, but because enzymes are proteins if the temperature exceeds 50? C the enzyme will be denatured and will no longer work. For this reason few cells can tolerate temperatures higher than approximately 45? C. Prediction: I predict that the breakdown of starch will be quicker when the temperature is increased until it exceeds 40? C. Then the diastase will no longer catalyse the breakdown of starch.. This is because amylase is made up of protein molecules and therefore it is easily denatured by heat, losing its lock and key (Ƴ) shape and no longer able to combine with the starch. I think the rate of breakdown of the starch will increase until this point because the increase in temperature will supply the molecules with more energy to react, and then it will slow down and the rate of reaction will be a lot longer. Dependant variable: rate of breakdown of starch, time
Independent variable: temperature of solution
Control variable: volume of starch solution
Volume of diastase solution
Concentration of amylase solution
Concentration of starch solution
Amount of iodine
Agitation
Introduction on diastase
Bernard Omolafe
Diastase is an enzyme that digests starch. It is most active at a normal pH (almost neutral), diastase comes from the Greek word diastases; A natural group of enzymes (Amylase and Amylase) that have the ability to break down starchy carbohydrates into simpler sugars acceptable to the yeast. Enzyme, having physiological, commercial, and historical significance, also called Amylase. 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. Set the water bath to 40? C
Procedure
Using a measuring cylinder measure 10ml of starch from the same solution each time
Pour 10ml of starch into one test tube
Pour 5ml of diastase into another test tube
Put the test tubes into the water bath
Put a thermometer into the starch solution
Put 2 drops of iodine into each part of the spotting tile
When the temperature becomes constant mix the diastase with the starch and start the stop clock at the same time
stir twice with a stirring rod for equal agitation
stir the mixture twice with the glass stirring rod
Every 30seconds remove a small portion of the solution and put into a spotting tile dimple with iodine in
Record the time that the solution doesn't change the colour of the iodine
Repeat this at a range of different temperatures
use ice to reach a lower temperatures
change the temperature of the water bath
Repeat each temperature twice to get more accurate results
Procedure - add 5cm of starch to each of two test tubes. After putting the diastase in a water trough through the different temperatures, add it to the starch solution. Then add iodine. Leave overnight for best results. See what colour it has turned to. temperature 14? C Time in minutes colour 1st experiment colour 2nd experiment
0 black black
0.30 black black
1 black black
1.30 black black
2 black black
2.30 black black
3 black black
3.30 black black
4 black black
4.30 black black
5 black black
5.30 black black
6 black black
6.30 black black
7 black black
7.30 black black
8 black black
8.30 black black
9 black black
9.30 black black
10 black/brown black/brown
10.30 black/brown black/brown
11 black/brown black/brown
11.30 black/brown brown
12 brown brown
But I will only do the most significant ones 25|c 40 and 60 My graph shows that I have no anomalous results and they all follow a smooth curve My graph shows that there is an optimum temperature . The first graph is a curve with all points in a place I would expect them to be.
OBSERVATION
As we can see, the blue- black solution representing the digestion of starch. All the others turned purple- black solution showing that the optimum is 40.
So it supports the theory that states that an enzymes function at an optimum of 40
As we can see from the graph it proves my hypothesis that the optimum of diastase as like most other enzymes function at 40 also we can notice that at 60 it begins to dysfunction and then denaturing occurs.
So therefore my hypothesis is true. Analysis.
My first graph shows the time taken for the amylase to break down the starch at a range of temperatures.
The graph is a curve showing that at 14? C the breakdown of starch to take 11.30 min and 37? C to be the optimum temperature taking 0.35 minutes to breakdown the starch, after this temperature the enzyme is denatured. The proteins structure unravels, which changes the shape of the molecule, and exposes the inner cells of the protein to the killing temperature. The gradient shows that the enzyme is not working as efficiently as it could and therefore needs a greater amount of time to breakdown the starch.
This supports my prediction that diastase works best at temperatures slightly below 40? C. My graph showing the rate of reaction between the starch and the amylase shows that as the temperature increases to 40? C the rate of reaction also increases and that as the temperature increases above 40? C the rate of reaction will decrease rapidly.
The peak in the rate of reaction is 37? C which is the best temperature for efficient use of diastase. The reason for the decrease in rate of reaction after 37? C is the enzyme is damaged.
CONCLUSION AND EVALUATION
In conclusion I think my experiment worked out well according to plan and I think this proves the theory beyond every reasonable doubt that Diastase is an enzyme like others exhibiting different effects under change in temperature.
Also that it has an optimum of 40 and denatures at temperatures exceeding that 40 which is the maximum and the highest the enzyme would function at
My results proved my prediction to be correct. The breakdown of starch is quicker as the temperature increases until the optimum temperature after which it begins to slow down.
I think my results were sufficient to support a firm conclusion, but if I had the chance to investigate further the effect of temperature on the breakdown of starch I would take more results focusing on the higher temperatures and try to pinpoint the optimum temperature.
