The presence of inhibitors affects the enzyme activity. Inhibitors are chemicals, which slow down the rate of enzyme activity. They do this by changing the shape of the active site. Inhibitors are categorised into two groups:
- Reverse inhibitors-they only bind temporally to the enzyme.
- Non-reversible inhibitors- it attaches itself permanently to the enzyme, this leaving the enzyme unable to carry out further catalysis reactions.
Enzyme- Amylase
There are many enzymes, but the one, which I’m concentrating on, is the enzyme amylase.
Amylase is the general name for an enzyme, which breaks down starch.
Starch makes up a large proportion of our diet and needs to be made soluble before it can be absorbed into our bodies. In order to carry out this chemical break down, various glands produce digestive juices containing amylase. Saliva contains one type of amylase and pancreatic juice contains another. Different amylase enzymes are produced by other organism including fungi and bacteria, which carry out external digestion. Any organisms that grow on starch must produce enzymes to break it down.
Hypothesis:
My prediction is that the more concentrated the amylase is, the larger the rings will be on the agar plate. I predict this as I believe that as the concentration of the enzyme is increased, the number of collisions will increase, this speeding up the reaction. This being all stated in the background research above.
For my second problem, I predict that as the temperature increases, the rate of reaction will also increase. This being due to the kinetic theory, which states that heat makes the molecules vibrate causing them to collide with each other.
Practical design plan:
Method for experiment 1:
I’ll begin my experiment by placing all the apparatus needed onto the work desk. I will arrange the test tubes in the test tube rack and begin by diluting the concentrations. 2ml of 2% amylase will be transferred into a measuring cylinder and mixed with 2ml of distilled water, this diluting the amylase concentration to 1%. 2ml of this solution will then be placed into a test tube and the remaining 2ml of the solution will be used for the dilution of the next concentration. The excess 1% amylase will be placed in the measuring cylinder and combined with 2ml of distilled water. This dilution creating a 0.5% concentration of amylase. 2ml of 0.5% concentration will be placed into the test tube and the remaining will be used for the next dilution.
This process will be repeated until the 0.25% and 0.125% concentration of amylase has been made.
After the concentrations have been made, I will begin by cutting three holes into each starch agar plate using a 10mm cork borer and tweezers for extra support.
I will lift the lid of the agar plate slowly and using a pipette place 0.125% of amylase into three holes. I will repeat this process and pipette the same concentration into another petri dish. I will have to make sure the concentration is filled to the surface of the agar dish.
This process will be repeated with each concentration until each concentration has been placed in a petri dish.
Two of the petri dishes will not contain any concentration; instead distilled water will be added. This being the controlled condition.
The 14 petri dishes will then be placed onto a tray and placed in the oven over night at a constant temperature of 37 C.
The tray will then be taken out of the oven the following day and the petri dish lid will slowly be lifted and flooded with iodine. The iodine should identify any enzyme activity, which have taken place. The lid of the petri dish will then be closed and the effect of enzyme activity will be drawn with permanent pen onto transparent paper. This will allow me to calculate the radius of each concentration, which will show me a comparison between each enzyme concentration.
In my first investigation I will be varying the concentration of amylase. The enzyme concentrations I will be using are 4%, 2%, 1%, 0.5%, 0.25% and 0.125%. I’ve decided to half the concentration each time as it will allow me to clearly see the difference between each concentration and the increase and decrease of enzyme activity. The enzyme will be placed in a mixture of soluble starch (1%) and agar (2.5%) which has been heated and then sterilised. It will then be poured into a sterilised petri dish and a small amount of universal indicator will be added when the jelly has set. It will be kept in the oven at a constant temperature of 37 C. It is vital that the temperature remains the same through out the experiment as this will have great impact on the results if not. This is because if the temperature is increased the amylase and starch molecules will begin to move faster due to the kinetic theory. I will be using 14 petri dishes; every two petri dishes will contain three holes, which have the same concentration of enzyme, this meaning I will have six trials, with each set of concentration. I decided to have six trials with each concentration as it would give me a wider set of reading and would show any patterns of trends clearly.
For my experiment a control condition is required. I will prepare a starch agar plate, but this time not adding an enzyme, but distilled water. This ensures that only amylase causes this affect with starch.
Method for experiment 2:
I will begin by placing all the apparatus on the desk. For this experiment I will only need 4 petri dishes (2 petri dishes at each temperature). I will gently open the lid of the petri dish and cut and remove three holes from the starch jelly using a 10mm cork borer and tweezers.
I will add 2% amylase to the hole, making sure it is filled to the rim of the starch jelly. The lid of the petri dishes will be closed and taken carefully to the fridge, which will be set at a constant 5 C, and two petri dishes will be kept at room temperature (23 C). I’ll measure the enzyme activity, the following day by flooding the petri dish with iodine and calculating the radius of the starch jelly, which has been broken down by the enzyme.
In my second experiment, I will be keeping the enzyme concentration constant (2% amylase concentration) but will be varying the temperature. I decided to use 2% amylase as I thought it would be more suitable and give a better view of results at different temperatures. I kept the concentration constant as varying both the temperature and concentration could affect my results. This would unable me to justify whether the concentration or temperature affected the enzyme activity. Two starch agar plates will be kept in the fridge at 5 C and two will be kept at room temperature (23 C). I will also be using the results in my previous experiment (experiment 1) where 2% amylase was used and kept in the oven at 30 C. The two starch agar plates will contain three holes and be placed at different temperatures. This meaning each temperature will have six trials each.
I will be varying the temperature, as it will allow me to see if enzyme activity differs as temperature increases.
As the temperature in this experiment is being varied, no controlled condition can be used. Placing the temperature at a set value is still regarded as a temperature. This making it difficult for me to have a controlled condition.
Materials:
The materials I will need for my investigations are:
4% Amylase: this will be the enzyme used to break down the starch.
Bottle of Distilled water: this will be used to dilute the amylase and will produce different concentrations. It will also be used to clean any contaminated equipment so that my results aren’t affected in any way.
Starch agar: this will test how reactive each enzyme concentration is.
Drops of Iodine: this will be used to identify any enzyme activity. It will turn the starch agar black/blue and show clear rings which will allow enzyme activity to be measured.
Apparatus:
The apparatus I’ll need to do my experiment are:
1 test tube rack-this will support and hold my test tubes.
1 measuring cylinder-to measure the volumes of any liquid substances used.
4 test tubes-I will need test tubes during the dilution of concentration. I’ll be doing this so that the amylase will be at the correct concentration for my investigation.
1 10mm cork borer-this will be used to cut three holes in each starch petri dish. The cork borer produces a well-rounded accurate shape and allows the jelly to be removed with not much mess. It also gives the same size cut for each hole, this preventing any errors in my results.
1 tweezers- this maybe used during the process of removing the jelly from the starch agar plate after it has been cut with a cork borer.
1 pipette-this will be used during the process of dilution when the liquids need to be transported from one test tube to another and when the concentration have to be placed into the starch agar hole.
18 petri dishes-these will contain the starch agar, which will be the base for the enzyme activation to occur. I will be monitoring the effects of amylase on the starch petri dish.
1 Tray-This will hold the starch agar plates in the oven, this prevented any spills of enzymes, which could alter my results.
1 Oven- I will use an oven to maintain the starch agar plates at a temperature above the room temperature. This is more accurate with an oven, as the temperature can be kept constant.
1 fridge-this will be used in my second experiment to maintain the starch agar plate at a temperature below room temperature. This allowing the starch agar to be kept at a constant temperature.
2 thermometer-this will be used to measure the temperature at which my starch agar plates are preserved at.
2 transparent sheets/permanent ink pen-these will be used to trace over the rings which discolour the agar plates after the iodine solution has been added. This will make it easier for me to measure the enzyme activity and produce greater accuracy.
Sources of error:
- Volumes of amylase have to be the same in each experiment as differences in volume would cause there to be a varied amount of molecules which could cause an alteration in my experiment. To avoid this I will fill the amylase to the surface of the petri dish making sure the hole is full.
- The reason for using the same oven for each petri dish is to make sure that are kept at a constant temperature. If they were placed at room temperature, the temperature would constantly be changing and therefore this not being a suitable environment for my enzymes.
- Each petri dish should be left at the same temperature for the exact same time, as leaving the petri dish in the environment for varied periods of time could make a large difference in the amount of enzyme activity taken place.
- During the dilution process I’ll have to make sure the amount of distilled water and amylase are equally halved to prevent any mistakes, I will use a pipette and measuring cylinder for extra accuracy.
I will be placing my results obtained by the experiment in a table:
Experiment 1
Experiment 2
I recorded all the relevant results obtained from my experiment and plot a graph from which I will hopefully be able to gain a strong conclusion. I will have six trials with each concentration as this will give a wider reading and show the pattern between each point clearly. I will finally evaluate the whole investigation and compare the results with the hypothesises I predicted.
The graph I will be plotting will be of concentration of amylase against the zone of discoloration (the area of starch, which has been broken down by amylase).
I will also be plotting another line graph showing temperature against rate of enzyme activity. The rings on the agar plate will be measures against the temperature.
Risk and safety assessment
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Lab coat: This is to protect clothing from becoming stained.
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Goggles: These will be used to protect the eyes. Must be worn especially while using iodine as it can be irritable to the eyes.
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Heat proof gloves: to protect my hands from the heat while taking the agar dishes out of the oven.
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Tie back loose clothing: this will prevent any spillages or glassware being broken.
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Tie back long hair: stops any spillage’s and won’t interfere with the experiment.
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Sterilise the agar plates: I will need to heat the starch agar plates as it could become contaminated and produce incorrect results.
- Only open the lid of the petri dish as far as necessary and close immediately as there could be a risk of inhaling the enzymes.
- Take care to keep the petri dish level on the bench and not to spill any enzyme as this could alter the results.
Method
Experiment 1:
All materials and equipment were placed on the lab worktable. Four test tubes were then arranged in the test tube rack and labelled (0.125%, 0.25%, 0.5%, 1%, 2%, 4%), this preventing any confusion.
An unmeasured amount of distilled water was placed in a beaker; this was used for cleaning the pipettes and made it more accurate and easier to transfer the distilled water, which was needed for dilution.
2ml of amylase was transferred using a pipette into the measuring cylinder and mixed with 2ml of distilled water. This creating a 4ml solution of 1% amylase.2ml of the solution was placed into the test tube labelled 1% and the remaining 2ml of solution was used in the next dilution.
The measuring cylinder was washed with distilled water making sure it was clean and no amylase had remained. The excess 2ml of 1% amylase was then placed into the measuring cylinder and mixed with 2ml of distilled water. This dilution creating a 0.5% concentration of amylase.
The 0.5% concentration of amylase was then placed into the test tube labelled 0.5% and the 2ml remaining was used in the next concentration to make an enzyme concentration of 0.25%. This process was repeated until 0.125% of amylase concentration had been made.
After the concentration had been (MADE), 14 starch agar petri dishes were collected and placed on the work surface. Slowly the lid of the petri dish was lifted and using a 10mm cork borer and tweezers the jelly in the dish was cut and removed, this left a well like hole in the jelly. In each agar plate three holes were cut and filled with a concentration.
4% amylase was taken from its bottle with a pipette and slowly squeezed into the hole, making sure it didn’t over flow and that it was filled to the surface of the starch agar. The dish lid was slowly placed onto the petri dish and was labelled 4%. As six trials were being used, two petri dishes were needed for each concentration. Therefore 4% amylase was again added in the same way to another petri dish. This process also occurred with the 2% amylase concentration.
The 1% amylase was however removed from the test tube and the process of filling the starch agar holes was repeated till each concentration was placed into petri dishes.
A controlled condition was used in this experiment and instead of an enzyme being placed in the agar plate, distilled water was lifted from the beaker using a pipette and gently squeezed into the six starch agar holes already cut using a 10mm cork borer and tweezers. It was then labelled and the 14 petri dishes were carefully submitted onto a tray and placed in the oven at 30 C.
The tray was taken out of the oven the following day and placed on the table careful ensuring no enzyme had spilt.
The weakest concentration, this being 0.125% was flooded with the iodine solution first. The petri dish had to be tilted; this ensuring the whole surface area of the starch agar plate had been covered.
The lid was closed slowly and rings of discoloration appeared on the jelly. To record the data, transparent paper was placed on the lid of the petri dish and the zones which showed the breakdown of starch were drawn with a permanent ink pen. The data was recorded and the area of enzyme activity was measured on mm graph square paper.
The technician then disposed of the starch agar plates, as they were no longer needed.
Experiment 2:
To start of this experiment four petri dishes were needed. The lid was slowly opened and three holes were removed from the petri dish using a 10mm cork borer and tweezers for extra support.
To each hole, 2% amylase was added using a pipette, making sure no enzyme concentration over flowed and that the hole was filled to the top, this preventing any errors.
This process was repeated till all four agar plates contained 2% amylase.
The lids on the petri dishes were closed gently and two were taken to the fridge and another two were kept at room temperature. Thermometers were also placed with the petri dishes, this allowing me to know the exact temperature at which the agar plates were preserved at.
The following day the two petri dishes were taken out the fridge and placed on the desk. The lid was slowly lifted and was flooded with iodine. The petri dishes needed to be tilted; this ensuring the starch plate was totally flooded. The lid was gently closed and using permanent pen the enzyme activity was recorded on transparent paper.
Using the thermometer I measured the temperature at which the petri dishes were kept at.
This same procedure was used with the petri dishes, which were kept at room temperature.
The area of enzyme activity on the transparent paper were counted on mm square graph paper, this allowing the results to be compared.
The agar plates were then disposed of by the technician.
