Increase in temperature
It is predicted that the rate of enzyme action will increase in proportion with the increase in temperature. The theory behind this prediction is known as the Kinetic Theory. This states that the higher the temperature is, the more collisions there will be between the substrate (fat) and the enzyme as a result of the increased energy of the enzyme. This means that as the temperature increases more enzyme will come into contact with more substrate and the reaction rate will therefore increase.
Optimum Rate
The enzyme action will reach an optimum rate at a certain temperature.
Higher Temperatures
After this point the rate of enzyme action will slow despite the further rise in temperature. This is because the heat above a certain point changes the shape of the active site of the enzyme so that the substrate no longer fits. At a certain temperature the enzyme action will stop all together as the enzyme becomes denatured. This was demonstrated in a previous experiment where the enzyme amylase, which is used to break down starch into sugar, was placed in a starch solution at 35 degrees Celsius. The enzyme was shown to break down the starch into sugar. A sample of enzyme was then boiled and then placed in an identical starch solution. At the end of the experiment this solution was found to contain starch but no sugar. This is because the enzyme had become denatured and was unable to break the starch down into sugar.
The lipase used is synthetically produced and may therefore be more stable at higher temperatures than predicted. The optimum temperature may be higher than predicted and the temperature at which it becomes denatured may be higher than predicted.
APPARATUS
Test tubes, test tube racks, water baths, beakers, thermometer, syringe, stop clock, stirring rod.
SAFETY
Laboratory rules were observed at all times.
PLAN
Two test tubes will be placed in water baths at the following temperatures: 10 degrees; 20 degrees; 30 degrees; 60 degrees and 80 degrees. Two test tubes will be left at room temperature. The average time taken for each of the two tubes at each temperature will then be taken. Each test tube will be left in the water bath for 3 minutes.
METHOD
In each of the 10 test tubes we added 3 cm3 of milk, 3 cm3 NaCO3, 5 drops of phenolphthalein and 1 cm3 bile salts. 1 cm3 of lipase enzyme was placed in each of the water baths and left to reach the temperature of the water. After the lipase had been in the water baths for 3 minutes it was added to each of the test tubes. The two test tubes being tested at room temperature also had 1 cm3 of lipase added. The time taken for the colour of the solution to change from pink to white was taken using a stop clock.
RESULTS
The results of the experiment are set out in the following table:
CONCLUSION & EVALUATION
At room temperature (22 degrees) the average time taken for the enzyme to digest all of the fat was 6 minutes 7 seconds.
At 10 degrees the average time taken for the enzyme to digest all of the fat was 14 minutes 3 seconds. This is therefore a decrease in the rate of enzyme action as the temperature decreases. This is in accordance with the prediction as enzyme action slows as temperatures decrease. This is because molecules move more slowly as temperature decreases so there will be fewer collisions between enzyme and substrate resulting in fewer reactions and a slower digestion time.
At 30 degrees the average time taken for the enzyme to digest all of the fat was 3 minutes 2 seconds. This is therefore a substantial increase in the rate of enzyme action from room temperature. This is in accordance with the prediction as enzyme action increases as temperatures increase. This is because molecules gain energy and move around more quickly resulting in more collisions and more reaction and a faster digression time.
At 60 degrees the average time taken for the enzyme to digest all of the fat was 24 minutes 2 seconds. This is therefore a substantial decrease in the rate of enzyme action from 30 degrees. This is again accordance with the prediction as enzyme action decreases as temperatures increase beyond a certain optimum temperature. The heat changes the shape of the enzyme so that the substrate no longer fits properly and the reaction slows.
At 80 degrees the enzyme did not digest any of the fat . The solution remained pink indefinitely. This because the shape of the enzyme had been changed by the heat so much that it was unable to bind with the substrate (the fat). The enzyme is said to be denatured. This is in accordance with the prediction as enzyme action was said to cease above a critical temperature. According to these results this critical temperature appears to be between 60 and 80 degrees.
According to these results the optimum temperature appears to be approximately 30 degrees as at this temperature the rate of enzyme action was fastest.
The bile salts were used to speed up the reaction. They physically break down the large fat molecules in the milk into smaller molecules which have a larger surface area so the lipase can chemically break down the fat molecules more easily.
The Phenol Phthalein was used because it is an indicator which is pink when alkaline and colourless when acidic. As a result of the presence of the NaCO3 the solution is alkaline at the start of the experiment. The indicator is therefore pink. As the enzyme acts the fat is broken down into fatty acids and glycerol. The solution therefore becomes more acidic and the indicator becomes colourless. The rate of enzyme action can therefore be monitored according to the speed of change in the colour of the indicator.
ACCURACY
The accuracy of the experiment may have been affected by the following factors:
Slight variations in the concentrations and volumes of enzyme, substrate and other substances used in each of the test tubes. This would be caused by inaccurate measurements of the substances.
Impurities in the solutions.
Temperatures may have fluctuated in test tubes as water baths not kept at a constant temperature.
To provide a more accuarate result the number of test tubes used in each temperature control could have been used. The average would then have been more reliable. More temperature controls could have been used (e.g. at 5 degree intervals). Natural lipase from the body could have been used in a separate experiment and the two experiments compared.