Enzymes are three dimensional proteins with a tertiary structure. They have an area on their surface called the active site which has a specific shape. The substance the enzyme acts on is the substrate. Both the substrate and active site have a specific complementary shape that allows them to bind together. When the substrate binds to the active site it forms a temporary bond and is know as an enzyme-substrate complex. The substrate reacts within the complex to form the products that then leaves the active site. This description of enzyme activity I know as the lock and key mechanism.
The induced fit theory with regards to enzyme activity is similar to the lock and key mechanism. However it states that when the substrate binds with the active site, the active site changes shape slightly so that the active site is fully complementary with the substrate.
The substrate sugars that I will use in my investigations will be either monosaccharide or disaccharide and this is an important difference as their shape will determine how easily they can bind to the active site of the yeast enzymes and thus how easy they can be broken down by the hydrolytic enzyme compared with glucose the most common respiratory substrate.
Below are descriptions of the sugar substrates that I will use in my investigations,
Glucose
Glucose is by far the most common carbohydrate and classified as a monosaccharide. It is the most common respiratory sugar and is directly phosphorylated in the first stage of respiration, Glycolysis.
Glucose is a six-carbon sugar. Its formula is C6H12O6 and its structure is shown below
Sucrose
Sucrose is a disaccharide and is made from glucose and fructose units: its structure is shown below,
The structure is easy to recognize because it contains the six member ring of glucose and the five member ring of fructose. Sucrose is used in many plants for transporting food reserves, often from the leaves to other parts of the plant.
Galactose
Galactose is one form of a disaccharide. It is a common sugar in milk and in yoghurt. Its structure is shown below
The chain form of galactose follows the same pattern as that for glucose, with 6 carbons.
Maltose
Maltose is a disaccharide and is made from to Glucose monomers. Maltose is a product of starch digestion and its structure is shown below
Fructose
Fructose is a monosaccharide and is a sugar found in honey. It too like glucose and galactose has a 6 sugar ring. Below is its structure
Lactose
Lactose is a disaccharide which consists of a glucose and Galactose molecule. It is found in mammalian milk, where it helps in giving energy to the newborn infant.
The shapes of these sugars are important in my investigation as they will determine which sugar produces the highest rate of respiration in yeast. The attachment of these molecules to the active site of the enzymes, and whether they are easier to break down by the enzyme will determine how fast the rate of respiration will be.
Prediction
I predict that both glucose and maltose will produce the highest rates of respiration in yeast compared with sucrose, fructose galactose and. This is because glucose is the main respiratory sugar in all species and has a shape that is full complementary with the enzymes involves in yeast respiration. Also glucose is the smallest from all the others sugars and so it will be easier to break down
Although maltose, which is made from two glucose molecules will have a slower rate of respiration than glucose I predict that it will have a faster respiration rate on yeast than the other sugars. This is because it is a disaccharide whereas glucose is a monosaccharide, so therefore it won’t be as complementary to the shape of the active sites of the enzymes involved in yeast respiration and so will be less readily broken down by the enzymes than glucose. Also because it is a disaccharide it will take time to be hydrolysed into monomers before it can start respiration.
I believe sucrose will have a slower respiration rate than Glucose and Maltose but a faster respiration rate then Fructose and Galactose. This is because it is made from Glucose and fructose and so because it contains glucose, the common respiratory substrate it will have a shape that is more specific to the active sites of the enzymes in yeast. Also because it is a disaccharide it will take time to be hydrolysed into monomers before it can start respiration
I expect Fructose, lactose and Galactose to be the slowest from the other sugars. This is because they are not common respiratory substrates and so they shape is not specific to the enzymes involved.
Null Hypothesis
My null hypothesis is that using different sugar substrates will not have an affect on the rate of respiration of yeast.
Method
Apparatus:
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100ml gas syringe- this will be used to collect the Co2 made by the yeast during the investigation. I will use the gas syringe as it is an accurate piece of equipment and will help to gain accurate results.
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Conical flask- this will be used to add the yeast and sugar, in order for the respiration of the yeast to take place.
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Rubber bung and pipe- this will be used to connect the conical flask and the gas cylinder together. Also the rubber bung will allow none of the Co2 to escape from the flask.
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Metal clamp and stand_ this will be used to safely secure the gas cylinder in place in order to use in my investigations.
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Heated Water bath- the conical flask will be placed in here. The water bath will allow the temperature to remain constant.
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25cm³ of 2% Glucose- This will be used as one my respiratory substrate.
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25cm³ of 2% Maltose- This will be used as one of my respiratory substrate.
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25 cm³ of 2% Sucrose- This will be used as one my respiratory substrates.
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25cm³ of 2% Galactose- This will be used as one my respiratory substrates.
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25cm³ of 2% Fructose- This will be used as one my respiratory substrates.
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25cm³ of 2% Lactose- This will be used as one my respiratory substrates.
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10g of yeast- This will be used to make a solution of yeast in order for it to be used in my investigations.
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10g in 200cm³ pH5 buffer solution- this will be used to form the solution of yeast.
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6*250cm³ beakers- this will be used to contain the yeast solution.
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Digital Balance- this will be used to accurately measure out the yeast in order to form the yeast solution.
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Weighing boats- these will be used to help measure out the yeast on the digital balance.
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Glass rod- this will used to stir the mixture of yeast and pH 5 buffer.
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Buffer tablet of pH 5- this will be used to maintain the pH of the distilled water to 6.
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Thermometer- this will be used to measure the temperature of the water bath for accuracy.
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Stop watch- this will be used to time my investigations.
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2 * 25 cm³ measuring cylinders- these will be used to measure out the sugar and yeast solution.
Step by step method
- Measure out 10 g of yeast using the digital balance and the weighing boats and place it into the 25cm³ beaker. Using the 250cm³ beaker, measure out 200 cm³ of distilled water and place the 10g of yeast into it. Place the buffer tablet into the solution and stir it until it dissolves. This will ensure that the yeast solution is at the optimum pH in order for the enzymes to work effectively. Using the glass rod gently stir the mixture to form the solution. This will ensure that all the yeast is dissolved into the water and that there is no solid present. Immediately after doing this place the beaker in the 35 cm³ water bath. This will allow the optimum temperature of the enzymes to be maintained in order for them to work effectively.
- Measure out 2g of each sugar using the digital balance and weighing boats and place them into separate 25 cm³ beakers. Measure out 100 cm³ of distilled water into 6 250cm³ beakers and place the weighed out sugars into each of them. Using a glass rod gently stir them and then immediately place them into the waterbath at 35³
- Leave the solutions of sugar and yeast in the waterbath for 15 minutes before carrying out the investigations. This will allow the solutions to be maintained at the correct temperature.
- After 15 minutes, using the two 25cm³ measuring cylinders, measure out 25cm³ of one of the sugars and 25cm³ of the yeast solution. As soon as they are measured out, pour them into the conical flask, put the rubber bung into the flask, this will prevent Carbon Dioxide from escaping to anywhere than the gas syringe. Start the stop clock immediately to avoid loss of time. Make sure the solutions are not left out for too long before the experiments as they temperature would drop and this will slow down the activity of enzymes and will affect your results.
- Continuously swirl the mixture. This will provide oxygen for the yeast and so will respire aerobically and this will prevent aerobic respiration from taking place.
- After every 30 seconds note down the amount of Carbon Dioxide produced. If, no more CO2 is produced after three concordant results then stop the investigation and move onto the next.
- Repeat the investigation for each sugar substrate five times. This will allow averages to be worked out and hence will provide more reliable results and a more reliable conclusion.
- I will then draw tables of raw results and average results. Using my average results I will draw graphs in order to find a gradient which will enable me to calculate the rate of respiration of each sugar. I will then calculate standard deviations. I will use this because it will measure the extent to which individual measurements vary around the mean. The greater the variation among the individual measurements, the bigger the standard deviation and vice versa.
I will then use the standard deviations to calculate the significance of the difference between two means via student T tests. I will carry out 3 student T tests.
Variables
The independent variable in my investigations will be the different sugars used. A different sugar will be added to the yeast and the rate at which it produces Co2 will then be calculated.
The dependent variable in my investigations will be the amount of CO2 produced by the yeast. I will measure the amount of CO2 produced by the yeast every 30 seconds for each sugar.
The Controlled variables in my investigation will be,
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The volume and concentration of sugar- I will always use 25cm³ of 2% sugar solution for each sugar. If I use a lower volume It means that there is not enough substrates to bind to the active site if the enzymes to cause a reaction. Higher concentrations will not affect the rate of respiration as the active sites of the enzymes will be all taken by substrates and so adding more substrate will not have an affect.
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The volume of yeast will also be kept constant- I will always use 25cm³ of the yeast solution. If I use too much then it means that there is lots of active sites of the enzymes available. Therefore more substrates will bind to them and thus making more enzyme-substrate complexes. This increase the rate of respiration. In order to carry out a fair test the same volume of yeast solution will be used as it would be unfair if it was not constant.
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The type of yeast- I will use Bakers yeast in all my investigations. Using different yeast will give me different results.
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Temperature- the waterbath that I will use will be kept at 35º and this will help to maintain the temperature of the yeast solution, sugar solution and the conical flask in which the sugar will be added to the yeast. A temperature of 35ºc is ideal as it is the optimum temperature for the yeast. If the temperature is too low then there is not enough kinetic energy given to the enzyme and substrate for it too collide and make a reaction occur. If the temperature is too high then the enzymes have lots of kinetic energy and begin to vibrate. This causes bonds in the enzyme to break and the shape of the active site of the enzymes changes. This means that no more substrate can bind to that active site and the enzyme becomes denatured. Before I carry out my experiment I will check the temperature of the waterbath by using a thermometer. This will be done for accuracy.
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pH- buffer tablets will be used to maintain the pH of the water used to make the solution. The buffer tablets that I will use will be pH 6 and this is ideal as it is the optimum pH for the yeast. If the pH is too high or too low then ionic and hydrogen bonds in the enzyme breaks, this cause the shape of the active site of the enzymes to change and means that no more substrate can bind to that active site. The enzyme therefore becomes denatured.
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Time at which the volume of CO2 produced will be measured- the volume of Co2 produced by the yeast will always be measured after 30 seconds until the yeast stops respiring.
Before carrying out my actual experiment I will firstly carry out a control test, to see the rate of respiration of boiled yeast using the same sugars and the same method as above. Below are the results of the test.
Observations, measurements and Precautions
- I will make sure that I use the procedure for all my experiments. This will ensure accuracy and reliability.
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Whilst carrying out my investigations I will make sure that I avoid making the parallax error. The parallax error occurs when the eye is not placed directly opposite a scale when a reading is being taken. Therefore I will make sure that when taking a reading I bend down so that I am parallel with the reading.
- I will make sure that I take regular measurements at fixed time intervals, after every 30 seconds for each of my experiments. This will help me maintain a fair test.
- I will also measure the temperature of the 35°c water bath before and after my investigations to make sure that the optimum temperature of the enzymes is maintained and to also ensure that I carry out a fair test.
- I will also measure the pH 5 buffer solution before and after my experiments to make sure that the optimum pH of the enzymes is maintained and to also ensure that I carry out a fair test
- Whilst carrying out my experiments I will make sure that I wash and dry all equipment used before proceeding with the next test. This will allow me to prevent any cross contamination that will affect my tests.
- I will make sure that the bung is fully compressed into the conical flask, so that each time the reading begins at zero. This will help me to avoid any errors whilst taking readings.
- If any spillage or accident occurs I will make sure that it is cleaned and washed straight away.
- I will make sure that I wear goggles and lab coat so that the substances used do not come into contact with eyes and skin.
- The glassware used will be handled with care and if an accident does occur I will make sure it is cleaned straight away.
- The sugar solutions that I will use are irritant. So if by accident it comes into contact with skin or eyes I will make sure that it is washed straight away.
- The ethanol produced by the yeast during anaerobic respiration is also irritant so again, if it comes into contact with eyes or skin I will make it is washed straight away.
- The yeast is not entirely dangerous however, if swallowed it can cause harm.
Results
Below are the results for all five tests I carried out.
Test 1
Test 2
Test 3
Test 4
Test 5
Average results
