Investigating the Effect of Temperature on the Activity of Protease Enzyme

Investigating the Effect of Temperature on the Activity of Protease Enzyme
Introduction The protease enzyme breaks down milk, which is insoluble and cloudy into amino acids, which are soluble and clear. In this investigation I will examine the effect that the variable of temperature has on the activity of the protease enzyme. Prediction I predict that as the operating temperature of the enzyme increases, the enzymes activity will increase at quite a fast rate from 0?C to around 30?C, and then continue to rise, at a much slower rate, until around 60ºC. The optimum temperature of the enzyme therefore will be approximately 60ºC. I predict that after 60? the activity of the enzyme will fall sharply. The reason for my prediction is that up to around 60?C the activity of the enzyme should increase because the heat gives the enzyme more energy meaning it is more active, however after 60?C the heat causes the enzymes active site to denature meaning it will show very limited activity. Plan In this experiment the manipulated variable will be the temperature at which the reaction between the protease enzyme and milk takes place.
The temperatures will be controlled by keeping the protease and milk solutions in water baths of different temperatures, both before and after they are added together. The range of values that will be tested are 10,20,30,40,50,60,70, and 80?C. We chose this range of values because our knowledge of protease enzyme is that it denatures at around 60?C. I will measure the results of these experiments using a piece of equipment called a colorimeter, because during our preliminary experiment we noticed that the liquids became clearer as the activity of the enzyme increased and suddenly cloudy again as the enzymes had denatured. It was suggested to our group after the preliminary experiment that light transmission could be measured using a colorimeter. A cuvette with a sample of the solution is placed into the colorimeter, which detects the amount of light that is transmitted by the solution and gives the result as a percentage. The Protease solution and the milk solution will be placed separately in the water bath at the correct temperature for 10 minutes, this is the amount of time which we found was needed during the preliminary experiment for the temperatures of the solutions to reach their operating temperature. After this time the two solutions were mixed together and placed back in the water bath for a further 4 minutes, allowing a minute for the result to be taken using the colorimeter. These timings are again based around the preliminary experiment when it took around 5 minutes to see a difference in the solutions at room temperature. To prove that it is indeed the enzyme and not the heat that breaks down the milk a control experiment will be carried out at each of the temperatures in the range we are testing, mixing only water with the milk and not the protease enzyme. To do the experiment our class was divided into 8 groups of 4 or 5 people, one group per temperature. We had a water bath set to the correct temperature for each of the tests. Each person took 5mls of 0.5% protease solution, and 5mls of 2% milk solution, measured with a syringe for accuracy, and heated them to their temperature in individual test tubes. The solutions were mixed together after 10 minutes, and allowed to react for a further 4 minutes, after 4 minutes the solution was transferred into a cuvette using a pipette. The cuvette is put into the colorimeter and the percentage of light transmission is measured and everyone noted down their results into a table for the entire class. We ensured that the experiment was a fair test by using a syringe to measure the amount of each solution we used, as for the result to be accurate the same amount of each solution should be used. We also carefully timed the 4 minutes that the solutions were allowed to react before we removed the protease and milk solution from the water bath and joined the queue for the colorimeter. Taking the average of 3 results per temperature will improve the accuracy and ensure that 1 anomalous result would not falsely manipulate the entire investigation. Results Temperature ºC % Of light transition of milk and protease enzyme after 5 minutes 2nd result 3rd result Transmission with water and milk Average light transmission through protease enzyme and milk 10 58.0 55.0 60.0 3 57.7 20 84.0 77.0 74.0 0 78.3 38 79.0 86.0 75.0 50 80.0 44 82.0 71.0 78.0 1 77.0 52 70.5 87.5 71.5 2 76.5 58 88 76 79 8 81.0 70 0.0 0.0 0.0 8 0.0 75 0.0 0.0 0.0 0 0.0 As you can see from the table some of the experiments were unfortunately not taken at the 10ºC intervals as planned. Analysis The graph shows the increase of the activity of the protease enzyme up to 59ºC, when a sharp fall to zero activity occurs. This is because the protease enzyme denatures at approximately 60ºC. The optimum temperature of the enzyme shown by the Graph is 59ºC, only slightly less then my prediction of 60ºC. The graph however peaks twice, 1st at about 30ºC, which is around the Human body temperature, the activity then falls by around 4% between 37ºC and 48ºand rises up to the optimum temperature of. I did not anticipate this, infact I expected that the increase in activity would slow after 30ºC but not fall. Evaluation The accuracy of the results for this investigation although good enough to roughly outline the activity of the enzyme, were greatly hindered by the fact that our whole class had to work with only one colorimeter to measure their results. Ideally we would have had a colorimeter per group but unfortunately the resources for this weren?t available. I believe that this could have been the cause of some anomalous results, for example the unusual dip in activity at 44ºC and 52ºC could have been caused by too little time for the reaction to occur, or by mistakes in the amounts of protease or milk measured and used in the experiment. The other anomalous result was the control experiment for 38ºC, in which water and milk solution had a 50% light transmission. I suspect that this experiment may have somehow become contaminated with the protease enzyme, which would have caused the unusually high reading. The experiment is very suitable for this investigation it provides accurate results as to the activity of the enzyme without manipulating it?s results. The experiment would have produced more accurate results if a measurement of activity had been taken at intervals of 5ºC, making the exact temperature at which the enzymes denature and the optimum temperature of the enzyme much clearer from the results. If we were to conduct this experiment again I would suggest that the different groups working on different temperatures should have been set off at 5 minute intervals which woud greatly reduce the waiting time to take the results of each experiment leading to greater accuracy throughout the results.

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