The ‘lock and key’ hypothesis can also be used to illustrate a denatured enzyme.
Denatured Enzyme
No chemical reaction can occur due to the different profiles of both the yeast enzyme and the glucose molecule.
Glucose has incorrect shape for the yeast enzyme’s active site and cannot therefore combine to complete the enzyme/substrate complex.
Variables:
Independent: The temperature at which the yeast enzymes are allowed to respire the glucose molecules.
I will use the following range of temperatures: 30, 35, 40, 45, 50, 55˚C.
I have selected to investigate these temperatures because I have predicted an optimum dough incubation temperature of 45˚C, and I will therefore test temperatures both lower and higher than my estimated one.
To maintain these temperatures, I will temperature equilibriate. This will be executed by allowing the temperature of the solution inside the test tube to equal that of the water in the water bath, before collecting any carbon dioxide gas given off.
Dependent: I am going to measure the volume of carbon dioxide gas collected in a gas burette after a controlled length of time, in cm³.
Control:
The size of the test tube.
If the test tube is large, then more carbon dioxide gas will be able to pass through the delivery tube and collect in the gas burette, during the controlled length of time.
The type, concentration and volume of yeast solution.
There will be the same number of yeast cells to respire the glucose each time. Also, it will certify that the only factor affecting the results is the temperature of the yeast solution, and not the concentration or type of yeast solution.
The time taken for carbon dioxide gas to collect in the gas burette.
This allows the results to be fairly evaluated (fair test).
The volume of water in the water bath.
This will ensure that the investigation is fair and again, it makes certain that this is not another influencing factor that affects the volume of carbon dioxide gas collected in the gas burette.
The number of times the test tube is shaken.
Shaking increases the chances of successful collisions between the glucose molecules and the yeast enzymes active sites.
The size of the gas burette.
If the gas burette is large then the volume of carbon dioxide collected will differ to that which has collected in a narrower one, because the calibrations will disagree, and the results would not be able to be compared fairly.
The size of the delivery tube.
If the delivery tube is longer, then the volume of carbon dioxide collected in the gas burette will not equal that collected from a shorter delivery tube, as less carbon dioxide will have been able to pass through the tube and collect in the gas burette in the controlled length of time, as there would have been if the tube was shorter.
Number of Replicates:
Each temperature will be repeated three times and a mean result for the six different temperatures will be calculated.
Replicating increases the reliability of average results and allows you to identify and discard any anomalous results.
Safety:
A Bunsen burner can easily burn you, so ensure that your hair is tied back and there are no flammable items close to the flame.
The tripod and gauze will become hot after heating, so allow them to cool and wear fireproof gloves when handling them both.
The water in the water bath will become warm when they are heated for the higher temperatures, so care should be taken not to spill any.
The yeast solution will likewise become moderately warm, and again, you should ensure that you don’t spill any onto your skin.
Finally, wear safety goggles throughout the investigation to prevent any warm water of solution to come into contact with your eyes.
Apparatus:
Two tripods
Two gauzes
Two thermometers
A large beaker
A small beaker
A Bunsen burner
Two fireproof mats
A plastic container
A clamp
A clamp stand
A calibrated gas burette
A bung
A delivery tube
A test tube
A measuring cylinder
A syringe
Yeast
Glucose
Yeast extract
400cm³ of water in the water bath
400cm³ of water in the plastic container
A top pan balance
A glass rod
Filter paper
Safety goggles
A stopwatch
Delivery tube Gas burette
Bung
Thermometer Beaker Clamp stand
Test tube
Water Water Clamp Plastic container
Yeast solution
Gauze
Tripod Bunsen burner
Fireproof mat
Preliminary Plan:
First, I will execute preliminary testing. This is necessary to discover if the controlled time for the carbon dioxide gas to collect in the gas burette is sufficient enough, to decide whether to use a suspension of yeast in glucose solution or dough, to find the best way to temperature equilibriate, to learn the best method by which to collect the volume of carbon dioxide gas given off in a controlled length of time, and to discover the range of temperatures I will use in the investigation.
I have chosen to time for two minutes, as I believe it to be an adequate time in which I will be able to obtain appropriate results. I will also use a yeast suspension because it is more practical than dough, as it would be both difficult and time consuming if I had to wash the greased measuring cylinders after each test, and it would also be complicated when measuring how much the dough had risen accurately. To temperature equilibriate, I will use a water bath, a Bunsen burner and a thermometer. Also, I will use a thermostatically controlled water bath to maintain the temperature inside the test tube.
To collect the carbon dioxide gas, I will use a gas burette instead of a gas syringe, as I do not believe that enough carbon dioxide gas will be released in two minutes to allow the gas syringe to work effectively, and thus, preventing me from obtaining sufficient results.
Preliminary Results:
From the above results, I can see that two minutes is a sufficient time length in which the carbon dioxide gas can collect. However, I will select a narrower range of temperatures. For instance, no gas collected has collected at 10 or 20˚C, so the lowest temperature I will investigate will be 30˚C. At 60˚C no carbon dioxide has collected either. Therefore, the highest temperature I will test will be 55˚C.
Plan:
Using a top pan balance, weigh a recipe for the yeast solution, containing 3g of glucose: 2g of dried yeast: 1g of yeast extract: 100cm³ of water.
Pour these ingredients into a small beaker and stir with a glass rod.
Set up the remaining apparatus, as shown in the apparatus list.
Using a syringe, measure 20ml of yeast solution and ensure that there are no air bubbles at the top of the syringe, which might otherwise prevent the measurement from being accurate.
Syringe this into a test tube.
Place the test tube into the thermostatically controlled water bath at the required first temperature.
Heat the large beaker containing water using a Bunsen burner to the first selected temperature.
Once both the yeast solution and the water bath are at the same temperature, place the test tube into the water bath and secure the bung.
Fill the gas burette with water, ensuring that there are no air bubbles that would affect the results.
Position the gas burette over the end of the delivery tube in the plastic container containing 400cm³ of water.
Begin timing for two minutes as soon as the first bubble of carbon dioxide gas appears.
Also, begin shaking the test tube in an upward and downwards motion, ensuring that you do so for the same number of times for each replicate.
Record your results in a table and plot a graph of the average volume of carbon dioxide gas collected against the temperature of each test.
Method:
I executed the investigation following the above plan.
Results:
Replicate 1:
Replicate 2:
Replicate 3:
Mean Results:
Graph Analysis:
From the graph I can see that 45˚C is the optimum temperature, which supports my prediction. I can also observe that very little carbon dioxide gas was collected below this temperature. From looking at the gradient between 30 and 35˚C, there is very little difference in the volume of carbon dioxide gas given off. The same can be said about the gradient between 35 and 40˚C. However, from 40˚C the gradient rises steeply, suggesting that the volume of carbon dioxide given off at 45˚C and that released at 40˚C has a great difference, thus supporting my prediction of an optimum temperature of approximately 45˚C.
Conclusion:
I proved that the optimum temperature for dough incubation prior to baking is 45˚C. As I predicted, my results show that any temperatures lower or higher than 45˚C (the optimum) produce less carbon dioxide.
Explanation of Conclusion:
The aim of the breadmaking is to produce dough that will rise easily and have properties required to make good bread for the consumer. In bread dough where the oxygen supply is limited, the yeast can only partially breakdown the sugar. Alcohol and carbon dioxide are produced in this process known as alcoholic fermentation.
Alcoholic Fermentation:
Glucose Ethanol + Carbon dioxide + Heat + 2 ATP
During fermentation each yeast cell forms a centre around which carbon dioxide bubbles form, as a waste product of alcoholic fermentation. The increase in dough size occurs as these cells fill with gas. At 40˚C, a considerable proportion of the carbon dioxide produced by the yeast is present in solution in the dough. As the dough temperature rises, carbon dioxide held in solution turns into a gas, and moves into existing gas cells. This expands these cells and overall the solubility of the gases is reduced. Heat changes liquids into gases by the process of evaporation and thus the alcohol produced evaporates.
After 40˚C, and until 50˚C, both the yeast enzymes and the glucose molecules had lots of kinetic energy. This kinetic energy enabled them to move around more and thus, cause more successful collisions between the yeast enzymes active sites and the glucose molecules. Due to these collisions, the yeast was able to respire the glucose and release the carbon dioxide. The production of this gas at such a fast rate instigated the higher volume of carbon dioxide gas collected in the gas burette at these temperatures, than at those lower than 40˚C.
Kinetic energy causes more successful collisions:
Yeast enzyme
Glucose molecule
Movement of glucose molecules
Movement of yeast enzymes
Dashed lines = chemical activity
From 30˚C to 40˚C, little carbon dioxide gas was produced as the low temperatures caused both the yeast enzymes and the glucose molecules to have a modest supply of kinetic energy. This meant that they were not able to move around as much and thus cause successful collisions between the yeast enzymes active sites and the glucose molecules. Consequently, less glucose could be respired by the yeast, and therefore, less carbon dioxide was released as a waste product of anaerobic fermentation.
Less kinetic energy causes less successful collisions:
Yeast enzyme
Glucose molecule
Movement of glucose molecules
Movement of yeast enzymes
Dashed lines = chemical activity
45˚C has been proven to be the optimum temperature, because it is neither too hot nor too cold for the yeast enzymes. If the temperature were any higher than this optimum, then the enzymes would no longer be able to maintain the original shape of their active site, and would become denatured. Furthermore, the glucose molecules would be unable to combine with the active sites to complete the enzyme/substrate complex, and therefore, less anaerobic respiration would occur, and consequently, less carbon dioxide gas would be released, which causes the bread to rise. This explains why less carbon dioxide was collected in the gas burette at both 50˚C and 55˚C, as the high temperatures had caused the yeast enzymes to become denatured.
Denatured Enzyme:
No chemical reaction can occur due to the different profiles of both the yeast enzyme and the glucose molecule.
Glucose has incorrect shape for the yeast enzyme’s active site and cannot therefore combine to complete the enzyme/substrate complex.
The temperatures below the optimum of 45˚C caused the yeast enzymes to respire less glucose and therefore release less carbon dioxide as a waste product of alcoholic fermentation, as neither the yeast enzymes nor the glucose molecules had sufficient kinetic energy, which as I have previously explained, causes less successful collisions between the active sites and the glucose molecules, due to the lack of movement from the limit of kinetic energy. Therefore, less glucose molecules were respired and thus, less carbon dioxide was released at these low temperatures.
As fermentation takes place the dough slowly changes from a rough dense mass lacking extensibility and with poor gas holding properties, into a smooth, extensible dough with good gas holding properties. Therefore, to achieve this desired dough, the baker will have to maintain a dough incubation temperature of 45˚C.
Evaluation:
This investigation is reliant on human judgement, and for this reason it is inaccurate. For example, when we read the calibrations on the gas burette we recorded to one decimal place, when perhaps we should have recorded to two decimal places, if the gas burettes has sufficient calibrations to allow us to do so. Also, by using a dropping pipette to measure the volume of water for instance, instead of a measuring cylinder we could have improved the accuracy of the volumes of the liquids used in the investigation. To perfect the accuracy of the experiment, we could make certain that there are no air bubbles present in either the syringe or the gas burette, which would otherwise cause the results to be erroneous. To prevent any carbon dioxide entering or escaping the test tube, we could use cling film, in the place of a bung, which would improve the accuracy further. When the test tube was being shaken, we were unable to control the number of ‘shakes’ or its power. Therefore, we could perhaps use a machine that would vibrate the test tube for a controlled number of times in the controlled length of time the carbon dioxide gas had to collect in the gas burette. The water bath was difficult to maintain at a constant temperature, as the Bunsen burner was not appropriate. We could therefore use a thermostatically controlled water bath for this, as well as using it to control the temperature of the test tube containing the yeast solution. Finally, this investigation was inaccurate because carbon dioxide is to some extent soluble in water, and so some may have been lost as it dissolved into the water, thus, decreasing the actual volume of carbon dioxide gas collected in the gas burette.
The investigation is relatively reliable because it was replicated three times and mean results for each different temperature were calculated, allowing us to eliminate any inaccuracies or anomalous results. However, by replicating perhaps five times, the reliability will increase further.
To support my conclusion, I could complete a further investigation based around the optimum I discovered in this investigation. For example, I could use a range of temperatures from 40˚C to 50˚C, with 1˚C intervals. This is because the optimum temperature could be between 40˚C to 45˚C or 45˚C to 50˚C.
To extend the enquiry, I could use dough instead of a yeast suspension. I could also vary the type of flour, the type and amount of sugar, the duration of the incubation process, or a combination of these factors.
