Rennin Enzyme Experiment
Investigating the effects of changing temperature on the activity of enzymes
Background information:
Renin is an enzyme that catalyses the coagulation of milk. It is found in the stomach of many animals and is used in making cheeses and junkets. It is found in the gastric juices or gastric mucosa of many mammals, including humans. In the human stomach, particularly those of infants, rennin works to curdle milk so that pepsin, another stomach enzyme, can further breakdown the proteins into absorbable amino acids called polypeptides.
The aim of this experiment is to investigate the effect of changing temperature on the activity of enzymes. After experimentation the optimum temperature for enzyme activity will be established and the effects …show more content…
of varing temperature will be identified.
Several experiments have already been conducted testing similar hypothesis and aims. All of these experiments also had very similar results. They found that approximately 37°C was the optimal temperature for rennin; it was at this temperature that the milk solidified quickest. Below that the reaction would occur far more slowly, sometime taking hours to complete, sometimes not reacting at all. Above 37°C, at approximately 45°C, the enzyme would become denatured and the reaction would never occur, even after the temperature was lowered back down to 37°C.1
Aim:
To investigate the reaction rate of the enzyme rennin at various temperatures
Hypothesis:
It is predicted that a rise in temperature (to approximately 40°C) will increase enzyme activity. Wth further increase of temperature the protein enzymes will denature, lose their shape and therefore decrease in activity.
Risk assessment:
Risk
Precaution
Burns from the hot water bath or hot plate
Ensure that all hot baths are set up in a visible area that is surrounded by minimal movement.
If burns occur run affected area under cold water for 5-10 minutes depending on severity. Major burns should seek medical assistance
Glass breakage can cause cuts/wounds
Use test tube rack to steady test tubes. If glass breakage occurs immediately alert teacher, sweep up broken glass using a broom or dust pan and dispose of it in the appropriate bin
Apparatus:
Equipment
Rationale
1 hot plate
Heats water in hot bath for raising temperatures of milk and rennin above 30°C
1 hot bath holds water which is heated by hot plate
1 ice bath (500mL beaker + 6-7 ice cubes)
Cools milk and rennin for testing temperatures below 30°C
3 regular test tubes
Holds the 20mLs of milk required for each trial
3 micro test tubes
Holds the small amount of rennin required
1 test tube rack
Holds regular sized test tubes in place so handling does not influence reaction
20mL of milk
Acts as substrate
2mL of rennin solution
Acts as enzyme for milk
1 large pipette
Gives precise measurment of milk
1 syringe
Gives precise measurement of rennin
2 thermometers
Measures temperature of milk and rennin when they are heated or cooled
1 timer
Measures time taken for milk to set
Method:
Constant Variables
Factor
Importance
Method of control
Volume of milk
The amount of milk determines the amount of substrate the enzyme has to work on which therefore effects the reaction rate.
Keeping a constant amount of milk for each trial. Use Pipette
Volume of rennin
The amount of enzymes determines the amount of chemical reactions possible to occur.
Keeping a constant amount of enzymes for each trial- 1ml per every 10ml of substrate. Use a syringe
Rennin and milk brought to the same testing temperature
Temperatures must be constant for both substances to ensure accuracy when they are mixed.
Place each substance in a hot or cold water bath with thermometers in their test tubes. Remove from water bath when the same temperature has been reached
Same time recorder
Timing must be accurate. With the same person reaction rate to press go and stop would be similar for each trial.
Have the same time recorder for every trial possible
Same setting standard
A setting standard must be determined to make timing of reaction rate accurate.
Make a class decision on what is classified as set
Standerdized thermometers
To ensure accurate temperature readings
Ensure all thermometers are standardized before experimentation
Type of milk- from the same container
Different types of milk may influence enzyme activity
Use the same milk for each trial
Type of junket- from the same container
Different types of rennin may influence enzyme activity
Use the same rennin for each trial
Whether the solutions should be stirred or not
A stirred or shaken solution may speed up reaction rate because more enzyme collisions would occur and faster
Make a class decision on whether to shake/stir mixture or keep it still
1.
Measure 20mls of milk using the pippette and release it into a regular test tube
2. Measure 2mls of rennin using the syringe and release it into a micro test tube
3. Place thermometers in both test tubes ensuring that the rennin does not overflow
4. Fill a 500ml beaker with 250ml of water and 6-7 ice cubes
5. Place both test tubes in the ice bath
6. Watch both thermometers until they reach 0°C
7. Pour the rennin solution into the test tube of milk. Begin timing as soon as all the rennin is poured into the milk
8. Shake the test tube slighlty to mix the rennin and milk together
9. Stop the timer once precipitate has formed or the milk has completely …show more content…
solidified
10. Repeat steps 1-9 using various other temperatures
Note: use a hot plate and hot water bath when testing temperatures above 30°C
Results:
Temperature (˚C)
Average time taken for milk to set (min)
0
No reaction – did not set
10
5min +
20
4:42
30
4:37
40
1.29
50
1.48
60
5min +
70
No reaction – did not set
80
No reaction – did not set
Discussion:
When temperatures were either very low (0-20˚C) or very high (70-80˚C) enzyme activity did not occur or was minimal. This is because cooler temperatures decrease the amount of kinetic energy within the enzyme molecules. If there is not a substantial amount of kinetic energy, enzyme molecules are unable to collide with their substrate which therefore prevents reaction from occurring. Because enzymes are proteins when temperatures were too high the enzymes denatured, lost their structure and shape, making their active sites no longer complementary to their substrate/s. 2At temperatures around 40-50 ̊C enzyme activity rapidly increased and the milk set under two minutes. This temperature range is therefore the optimum temperature for enzyme activity because enzymes obtain substantial amounts of kinetic energy and do not become denatured.3
Accuracy was not constant throughout the experiment as small amounts of rennin were lost everytime a thermometer was placed in the micro test tube.
Some milk was also lost when the rennin was added to it and the test tube was shaken. This may have affected the reaction rate of the enzymes. It was very difficult to calculate the exact temperature at which the enzyme and substrate were mixed because their temperatures dropped or rose rapidly when they were removed from the hot or cold baths. This further affected the accuracy of the experiment as temperatures tested were not exact. Repeated trials of the same temperature all had similar results which made the averages precise. There were no outliers so averages were not too high or too low. If an outlier occurred during experimentation because of known reasons the trial was tested again. Each temperature was tested three times making the experiment reasonably reliable however different groups tested each temperature which may have affected the reaction times recorded because of different perceptions about setting points and the speed of the persons timing. The limitations of this experiment were therefore caused by human
error
To prevent errors during experimentation more caution should have been taken when: mixing and shaking substances, recording the reaction rate, interpretation of setting time and the handling of test tubes. When shaking and handling the test tubes a stopper should have been used to prevent loss of mixture and exposure of heat from the person’s hands. Timing should have been conducted by the same person for every trial however this would have taken far too long. Interpretation of setting time should have been clearly explained or demonstrated before conducting the experiment.
The results of this experiment correlated closely to research undertaken about the effects of changing temperature on enzymes. This therefore made the practical more valid and reliable. Most constant variables were followed and monitored precisely which further added to the fairness of experimentation. A few constant variables that may have been affected by human error were the temperatures at which the substances were brought to, the volume of rennin and the perceptions of the milks setting point, although the same instructions were given to all students.
Conclusion:
Enzyme activity therfore increases as temperatures increase to their optimum temperature (37-45°C). Once temperatures exceed the optimum temperature the enzymes denature (lose their shape) and become inactive.
Bibliography
Jirage, R 2009 Function of Rennin Enzymes, Ella Bache College, accessed 3 November 2013, .
Brooklyn College, n.d. Effect of temperature on enzyme activity, City University of New York, accessed 3 November 2013, .
Chidrawi, G, Robson, M & Hollis, S 2010, Biology in Focus, 1st edn, Cengage Learning, Australia
Background information:
Renin is an enzyme that catalyses the coagulation of milk. It is found in the stomach of many animals and is used in making cheeses and junkets. It is found in the gastric juices or gastric mucosa of many mammals, including humans. In the human stomach, particularly those of infants, rennin works to curdle milk so that pepsin, another stomach enzyme, can further breakdown the proteins into absorbable amino acids called polypeptides.
The aim of this experiment is to investigate the effect of changing temperature on the activity of enzymes. After experimentation the optimum temperature for enzyme activity will be established and the effects …show more content…
of varing temperature will be identified.
Several experiments have already been conducted testing similar hypothesis and aims. All of these experiments also had very similar results. They found that approximately 37°C was the optimal temperature for rennin; it was at this temperature that the milk solidified quickest. Below that the reaction would occur far more slowly, sometime taking hours to complete, sometimes not reacting at all. Above 37°C, at approximately 45°C, the enzyme would become denatured and the reaction would never occur, even after the temperature was lowered back down to 37°C.1
Aim:
To investigate the reaction rate of the enzyme rennin at various temperatures
Hypothesis:
It is predicted that a rise in temperature (to approximately 40°C) will increase enzyme activity. Wth further increase of temperature the protein enzymes will denature, lose their shape and therefore decrease in activity.
Risk assessment:
Risk
Precaution
Burns from the hot water bath or hot plate
Ensure that all hot baths are set up in a visible area that is surrounded by minimal movement.
If burns occur run affected area under cold water for 5-10 minutes depending on severity. Major burns should seek medical assistance
Glass breakage can cause cuts/wounds
Use test tube rack to steady test tubes. If glass breakage occurs immediately alert teacher, sweep up broken glass using a broom or dust pan and dispose of it in the appropriate bin
Apparatus:
Equipment
Rationale
1 hot plate
Heats water in hot bath for raising temperatures of milk and rennin above 30°C
1 hot bath holds water which is heated by hot plate
1 ice bath (500mL beaker + 6-7 ice cubes)
Cools milk and rennin for testing temperatures below 30°C
3 regular test tubes
Holds the 20mLs of milk required for each trial
3 micro test tubes
Holds the small amount of rennin required
1 test tube rack
Holds regular sized test tubes in place so handling does not influence reaction
20mL of milk
Acts as substrate
2mL of rennin solution
Acts as enzyme for milk
1 large pipette
Gives precise measurment of milk
1 syringe
Gives precise measurement of rennin
2 thermometers
Measures temperature of milk and rennin when they are heated or cooled
1 timer
Measures time taken for milk to set
Method:
Constant Variables
Factor
Importance
Method of control
Volume of milk
The amount of milk determines the amount of substrate the enzyme has to work on which therefore effects the reaction rate.
Keeping a constant amount of milk for each trial. Use Pipette
Volume of rennin
The amount of enzymes determines the amount of chemical reactions possible to occur.
Keeping a constant amount of enzymes for each trial- 1ml per every 10ml of substrate. Use a syringe
Rennin and milk brought to the same testing temperature
Temperatures must be constant for both substances to ensure accuracy when they are mixed.
Place each substance in a hot or cold water bath with thermometers in their test tubes. Remove from water bath when the same temperature has been reached
Same time recorder
Timing must be accurate. With the same person reaction rate to press go and stop would be similar for each trial.
Have the same time recorder for every trial possible
Same setting standard
A setting standard must be determined to make timing of reaction rate accurate.
Make a class decision on what is classified as set
Standerdized thermometers
To ensure accurate temperature readings
Ensure all thermometers are standardized before experimentation
Type of milk- from the same container
Different types of milk may influence enzyme activity
Use the same milk for each trial
Type of junket- from the same container
Different types of rennin may influence enzyme activity
Use the same rennin for each trial
Whether the solutions should be stirred or not
A stirred or shaken solution may speed up reaction rate because more enzyme collisions would occur and faster
Make a class decision on whether to shake/stir mixture or keep it still
1.
Measure 20mls of milk using the pippette and release it into a regular test tube
2. Measure 2mls of rennin using the syringe and release it into a micro test tube
3. Place thermometers in both test tubes ensuring that the rennin does not overflow
4. Fill a 500ml beaker with 250ml of water and 6-7 ice cubes
5. Place both test tubes in the ice bath
6. Watch both thermometers until they reach 0°C
7. Pour the rennin solution into the test tube of milk. Begin timing as soon as all the rennin is poured into the milk
8. Shake the test tube slighlty to mix the rennin and milk together
9. Stop the timer once precipitate has formed or the milk has completely …show more content…
solidified
10. Repeat steps 1-9 using various other temperatures
Note: use a hot plate and hot water bath when testing temperatures above 30°C
Results:
Temperature (˚C)
Average time taken for milk to set (min)
0
No reaction – did not set
10
5min +
20
4:42
30
4:37
40
1.29
50
1.48
60
5min +
70
No reaction – did not set
80
No reaction – did not set
Discussion:
When temperatures were either very low (0-20˚C) or very high (70-80˚C) enzyme activity did not occur or was minimal. This is because cooler temperatures decrease the amount of kinetic energy within the enzyme molecules. If there is not a substantial amount of kinetic energy, enzyme molecules are unable to collide with their substrate which therefore prevents reaction from occurring. Because enzymes are proteins when temperatures were too high the enzymes denatured, lost their structure and shape, making their active sites no longer complementary to their substrate/s. 2At temperatures around 40-50 ̊C enzyme activity rapidly increased and the milk set under two minutes. This temperature range is therefore the optimum temperature for enzyme activity because enzymes obtain substantial amounts of kinetic energy and do not become denatured.3
Accuracy was not constant throughout the experiment as small amounts of rennin were lost everytime a thermometer was placed in the micro test tube.
Some milk was also lost when the rennin was added to it and the test tube was shaken. This may have affected the reaction rate of the enzymes. It was very difficult to calculate the exact temperature at which the enzyme and substrate were mixed because their temperatures dropped or rose rapidly when they were removed from the hot or cold baths. This further affected the accuracy of the experiment as temperatures tested were not exact. Repeated trials of the same temperature all had similar results which made the averages precise. There were no outliers so averages were not too high or too low. If an outlier occurred during experimentation because of known reasons the trial was tested again. Each temperature was tested three times making the experiment reasonably reliable however different groups tested each temperature which may have affected the reaction times recorded because of different perceptions about setting points and the speed of the persons timing. The limitations of this experiment were therefore caused by human
error
To prevent errors during experimentation more caution should have been taken when: mixing and shaking substances, recording the reaction rate, interpretation of setting time and the handling of test tubes. When shaking and handling the test tubes a stopper should have been used to prevent loss of mixture and exposure of heat from the person’s hands. Timing should have been conducted by the same person for every trial however this would have taken far too long. Interpretation of setting time should have been clearly explained or demonstrated before conducting the experiment.
The results of this experiment correlated closely to research undertaken about the effects of changing temperature on enzymes. This therefore made the practical more valid and reliable. Most constant variables were followed and monitored precisely which further added to the fairness of experimentation. A few constant variables that may have been affected by human error were the temperatures at which the substances were brought to, the volume of rennin and the perceptions of the milks setting point, although the same instructions were given to all students.
Conclusion:
Enzyme activity therfore increases as temperatures increase to their optimum temperature (37-45°C). Once temperatures exceed the optimum temperature the enzymes denature (lose their shape) and become inactive.
Bibliography
Jirage, R 2009 Function of Rennin Enzymes, Ella Bache College, accessed 3 November 2013, .
Brooklyn College, n.d. Effect of temperature on enzyme activity, City University of New York, accessed 3 November 2013, .
Chidrawi, G, Robson, M & Hollis, S 2010, Biology in Focus, 1st edn, Cengage Learning, Australia