I am going to consider the effect of temperature on the activity of immobilised lipase and free lipase. When doing this I will need to consider the temperature of the treatment for each enzyme. If there are some differences between each of the lipases I am going to test, it could be due to differences in the preparation of each one.
Outline Method
An indicator can be used to show up the digestion of fats. When lipase acts on milk fats, the fatty acids produced react with alkaline sodium carbonate and make the solution more acidic (below pH 8.3). When the digestion of fats produces enough acid to bring the pH below 8.3 the phenolphthalein changes from pink to white. This is obvious result but it is not always easy to tell when all the pink colour has disappeared
Key Variables
The lipase and sodium carbonate concentration must be the same for all the experiments.
The same type of the milk will be use in all the experiments.
The same concentration and size of drops of immobilised enzymes and phenolphthalein will be used each time.
The end point will be judged against some untreated milk.
Method
First I will take 4 test tubes. I will measure 1cm3 of 5% lipase solution and 1cm3 of distilled water with pipettes and place this in a clean test tube. In another two test tubes I will measure 5cm3 of milk, 7cm3 of sodium carbonate and five equal drops of phenolphthalein. Then I will prepare the immobilised enzymes. I will make up 50cm3 of 3% sodium alginate solution in a beaker and add 50cm3 of 5% lipase solution. I will stir the mixture with a glass rod until the enzyme is evenly dispersed. Then I will draw 2cm3 sodium alginate/lipase solution into a clean syringe. I will pipette this, drop by drop, into the calcium chloride solution to form beads. I will recover all the beads with a spoon, transfer them into an empty beaker and wash them with distilled water. Finally transferring the beads into a clean test tube. I will then place all 4 test tubes in a water bath at 20°C for five minutes. I will check the temperature of the solutions with a thermometer.
As soon as the 5 minutes has completed I will mix them together and start a stopwatch. I will then record the time when the solutions turn white (like the untreated milk)
I will repeat this experiment at different temperatures (30, 40, 50, 60, 70°C). I will repeat each reading.
Risk Assessment
Lipase enzymes
This is in powdered form. All enzymes have biological activity and need to be treated with care. Avoid inhaling powder.
Sodium Carbonate and Phenolphthalein
Very low risk
Keep in clearly labelled container
Results
To enable me to compare the effects of temperature on each enzyme I calculated the averages of the times and then the overall rate of reaction for each using the formula:
1
time taken for colour change (s)
I then expressed the rate of each reaction as a percentage of the maximum rate.
The results are shown in the following summary table.
Summary results table
Conclusions
Main trends and patterns
The results show that there is a significant difference between free lipase and immobilised lipase, and their response to different temperatures. The raw data shows that immobilised lipase is less active in these conditions compared with free lipase.
Free lipase retains over 50% of its activity between 30 and 50°C. Above this temperature free lipase activity rapidly decrease to 0% (denatured), unlike at 20°C where it retained some activity.
On the other hand, it can be seen from the graph that the immobilised lipase retained over 40% of its activity between 30 and 50°C. It could be said therefore that the immobilised enzyme was more sensitive to the temperature but although activity was less it was still active at 60°C. There is an obvious anomaly at 50°C as the original and repeat time are too far apart, although it was interesting to see that the differences between the original and repeat time increased as the temperature increased. This may be an important trend or experimental error, which would need further investigation. This anomaly or any other experimental errors could have a significant effect on the final results when converted to percentages.
Explanation of results
Enzymes are globular proteins and have complex tertiary and sometimes quaternary structures. In the proteins polypeptide chains are folded into spherical (globular) shapes. Within these large molecules is found an active site, which is the part responsible for the functioning of the enzyme. Anything that affects the 3-D shape of a protein molecule affects its ability to function. In large proteins this 3-D shape is held in place by hydrogen bands and sulphur bridges. Increases in temperature make it more likely that these cross links become broken and as the polypeptide chains unfold, it is more likely the change in shape will affect the active site and stop the enzyme working.
The active site of enzymes acting on the same substrate must be very similar it is possible that variations in the preparations of enzymes can effect how quickly the enzyme substrate complex can form. It is therefore possible that the effect of increasing the temperature can be different.
In this case if the results for immobilised lipase were shown to be correct it would be possible to say that as the temperature increased some cross links were broken, which slowed the rate of reaction but other bonds that remained intact because of entrapment kept its activity over a wider temperature range. The effect of increasing the temperature further meant that the cross-links were broken despite being immobilised.
In the case of free lipase the cross-links could resist up to 60°C but at this point many similar links were broken and the activity fell quickly. It would be possible to investigate this idea by analysis of the preparation of each enzyme.
Experimental limitations
The most important limitation to this experiment is the difficulty in making an accurate judgement of the final end point. The colour of the phenolphthalein changes slowly in some cases. In the slower reactions this was particularly difficult. Whilst I made an effort to distinguish between the untreated milk and the reactions, and those where the solution remained pink by counting the rate of reaction as zero where there was no sign of change this remained quite an inaccurate method.
The other major limitation involved temperature treatment especially at higher temperatures it could have easily made a difference in the results. The solution itself took longer to reach these high temperatures so the time of treatment did vary.
The overall reliability of the investigation was also dependent on the time constraints. To cover the full range of temperatures there was only enough time to carry out one set of repeats. It is obvious from some of the variability of the results that this was an important limitation.
Overall it appeared that this investigation did provide some evidence to support my hypothesis that immobilised lipase would work better over a wider temperature range than free lipase.
Clearly repeating these tests at least twice would be the most important type of further work, which would be needed. If the trends shown in the first set of data were consistent it would also be important to investigate smaller increases in temperature between 40 and 60°C. Also seeing how the length of each temperature treatment affects the enzymes.
If time were available it would also be profitable to investigate a wider range of enzymes when immobilised by entrapment in a sodium alginate gel.
