Investigating the changing effects of temperature

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 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 370C was the optimal temperature for r ennin; 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 370C, at approximately 450C, the enzyme would become enatured and the reaction would never occur, even after the temperature was lowered back down to 370C. 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 400C) 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 c uts/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 300C 1 hot bath holds water which is heated by not plate 1 ice bath (500mL beaker + 6-7 ice cubes) Cools milk and rennin for testing temperatures below 300C 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 Met hod: 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- Iml 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 d etermined 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 activityUse 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 2 50ml of water and 6-7 ice cubes 5. Place both test tubes in the ice bath 6.Watch both thermometers until they reach OOC 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 solidified 10. Repeat steps 1-9 using various other temperatures Note: use a hot plate and hot water bath when testing temperatures above 300C Results: Temperature ( C) Average time taken for milk to set (min) No reaction – did not set 10 5min + 20 4:42 4:37 1. 29 50 1. 48 70 Discussion: When temperatures were either very low or very high 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. At temperatures around 40-50C 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.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 t which the enzyme and substrate were mixed because their temperatures dropped or rose rapidly when they were removed from the hot or cold baths. T his 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 ave 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 mixt ure and exposure of heat from the person's hands. Timing should have been conducted by the same person for every trial however this would ave 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, lthough the same instructions were given to all students . Conclusion: Enzyme activity therfore increases as temperatures increase to their optimum temperature (37-450C). Once temperatures exceed the optimum temperat ure the enzymes denature (lose their shape) and become inactive.