A factor effecting enzyme activity is the enzyme concentration. This relates to my hypothesis as the same equation applies when using the volume of an enzyme.
As the volume (or the concentration) of the enzyme increases the number of active sites are also increased, providing there is an excess of substrate molecules. An increase in active sites means that there will be enough sites for each substrate to be broken down. This saves time and it should, in theory speed up the reaction.
Experiment materials and apparatus
Enzyme Lactase: five different volumes - 6 mls, 5 mls, 4 mls, 3 mls and 2 mls (these will be needed three times for repeated experiment)
Milk (in which contains Lactose)- 5x 1 ml measures will be tested with each volume of Lactase.
Benedict solution (a test for sugar)
Stop Watch
1 Glass beaker, 3 plastic beakers(medium size)
Thermometer
Test tubes
Test tube Holders and forceps
Measuring cylinders
Pipettes
Bunsen Burner, Safety mat, Tripod, Clay pipe triangle
Safety Goggles
Method
Firstly I will put on safety goggles and then set up a safe water bath, using the Bunsen burner, tripod, clay triangle and safety mat. I will then half fill the glass beaker with water and heat to 100 degrees (boiling point). Next I will measure out the first volume of the Lactase (6ml) and 1ml of milk (this measurement will remain the same throughout the experiment) using the measuring cylinders, and heat separately in test tubes in a water bath of 40 degrees (an enzyme works its best at this temperature) in one of the plastic beakers. I will use the other two plastic beakers to keep hot and cold water, so to keep 40 degrees at a constant and also measure this temperature using the thermometer.
Using other test tubes I will place 5 drops of Benedict solution (using a pipette) into each test-tube. Depending on the time it takes the lactose to break down a specific amount of test tubes can not be set but 10 shall be used to start with, adding more if the lactose takes longer to break down. I will then place these test tubes into the holders to prevent spillage.
I will then mix the enzyme and the milk into the same test tube and replace in the water bath of 40 degrees and start the stop watch.
Starting at 0 seconds I will start the stop watch and then place 5 drops of the enzyme and milk mixture (so to keep the volume the same as the Benedict solution) into one of the test tubes holding the Benedict solution and place immediately into the 100 degree water bath. I will then wait to see what colour the solution will turn. When Benedict solution detects sugar it must be at a 100 degree temperature and it also will turn a green/brown colour. I will wait for a colour to become visible then record the result at 0 seconds. Then when it comes to 2 minutes I will repeat this test and continue every two minutes until the solution has turned a brick red colour, this will mean there is no lactose in the milk present.
I will repeat this procedure with the other enzyme volumes. If my hypothesis is correct then the 6ml volume of enzyme should take the shortest amount of time to break down. This is because in comparison to the 1ml of milk it is a larger volume and there will be plenty of active sites to bind with. The one which should take the longest is the 2ml of enzyme as this will have the least amount of active sites.
To keep this experiment fair I will take the following precautions:
- Keep safety goggles on at all times
- Keep hair tied back if it is long
- Place the Bunsen burner and 100 degree water bath in a safe place where it can not be knocked over.
- When lighting the Bunsen burner keep it on a safety flame so others can see it is on.
- Use the safety mat under the Bunsen burner and be careful not to touch any of the equipment that is being heated by it.
- Use the forceps to place the test tubes in and out of the 100 degree water bath.
- Whilst the test tube is in the 100 degree water bath place it away from the eyes as it may overheat.
- Handle the test tubes with care as they can easily break and place in a test tube holder.
- Do not drink any of the solutions in this experiment or drink/eat anything whilst carrying the experiment out.
- Keep all bags and coats out of the way as people may trip over them.
- Use a water bath at all times when testing the Benedict solution, as placing test tubes over a direct flame will crack and break the test tube.
- Make sure the water bath boiling over the Bunsen is only filled halfway as it could over boil. and cause spillage and burns.
- Keep all equipment in a safe place where spillage's will not occur.
To keep this experiment fair I will do the following:
- Keep the measurements of milk to 1ml at all times
- Keep the measurements of the Benedict solution and the solution of enzyme and milk the same at all times.
- Keep both water baths at their constant temperatures (40 degrees and 100 degrees)
- Test all solutions at 2 minute intervals
- Repeat the experiment at least twice more to give a overall average.
- Keep the concentrations of the enzyme the same throughout the experiment.
Results
Analysing
This graph is to show three repeated readings of my experiment, and shows for what length of time the enzyme lactase took to break down the lactose in the milk.
From what I can see the 2nd reading is the most different of the readings. The first three results for the volumes seem completely different to the ones in the 1st and second reading. But towards the end they seem to match up. To explain this I would say that one of the variables may have changed i.e. the temperature of the water bath (40 degrees) and this may have caused the enzyme to act quicker than the rest. Another possible explanation is that during the second reading the Benedict solution used may have been different and may have given false colour results.
To take an average I would say that even though the second reading seems to have been altered in some way the results for the 6ml volume of enzyme is still the fastest at breaking the lactose present in the milk down. Which supports my original hypothesis that 6mls of lactase will have a significantly higher amount of active sites and will speed up the process of breaking down the lactose.
The last two volumes, 3mls and 4mls of lactase are, as I predicted, the slowest at breaking down the milk. This is due to the fact that there were less active sites for the substrate to bind with and so the substrate must wait its turn to be broken down, this wastes time.
Evaluation
Although my results vary at points they still give the same implication, that 6mls of lactase is more sufficient for breaking down 1ml of milk in the shortest time, these results prove that my hypothesis was correct. This investigation and the conclusion of it may be used to benefit lactose intolerent people as they will not have to wait such a long length of time to get milk suitable for them to drink. Instead of using the normal dose of lactase , used by the lactose intolerant user, it could be possible to double or even triple the amount of lactose broken down, making it more suitable for them to use.
In future, if I ever repeat this investigation, things I could change to make this test more reliable are:
- To use the same Benedict solution for all readings
- Maybe use an electric water bath where the water would be a constant temperature of 40 degrees. I feel this would be useful as it was hard to read results and check the temperature was at a constant.
This investigation worked at a reasonable level, the fact that all my readings were not similar made it slightly less successful than I would have hoped, but the hypothesis was still proved right.
