Alevel Biology Coursework Enzyme Action

  • Hi,
    I did an experiment in school last week investigating the effect of temperature on both free and immobilized lipase. We used temperatures of 40 and 80 degrees C. At 40, both were active but at 80 only the immobilized lipase was active. However, its activity was greatly reduced to that at 40. I understand that the immobilisation process makes the enzymes more stable but why is this and why, if it is more stable, is the activity reduced at all at a higher temperature? In other words, what is the explanation for the decrease in activity in immobilised lipase between 40 and 80 degrees C when with free lipase the enzyme is simply denatured and activity is completely halted?
    Any ideas would be greatly appreciated!! Thanks

  • The immobilisation process makes the enzyme MORE stable, not completely stable. So, the activity of the immobilized enzyme will be greater than that of the free enzyme. Basically, just because its more stable it does not mean that it wont have reduced activity at certain temperatures.

  • The alginate beads (I presume) stabilise the bonds within the tertiary structure of the immobilised lipase, thus makes them more stable. However, SOME of the immobilised lipase will still denature, and so there is less activity at 80 degrees. The lipase which does not denature is still active and so can still hydrolyse whatever substrate it may be, so there is still some activity.

  • Overall, I believe my experiment went well and I believe that I gained sufficient results because I repeated each concentration 3 times, and investigated 8 concentrations in total. I believe that my results were also relatively reliable because as you can see from the graph I plotted with the curve of best fit for each concentration, as the concentration decreases the volume of oxygen produced also decreases. For example, the 100% concentration of hydrogen peroxide evolved a final average volume of gas of 77cm3 of oxygen while the 90% concentration evolved a final average volume of 73.3cm3. Also most of the points were on or close to the curve of best fit for each concentration. However there are some factors that I must take into consideration.

    Firstly, there were limitations of the apparatus that I used. Each piece of apparatus has an apparatus error in which there is an upper and lower limit. For example the balance had an apparatus error of ±0.01 which means that since I used 0.2g of yeast, this value could either be 0.21g or 0.19g. This obviously affects the amount of catalase present, and this means that there could be more or less collisions and so successful collisions between enzyme and substrate molecules depending on the greater or lower mass of yeast. For example, if there were more molecules of yeast the rate of reaction would increase because there would be more collisions between enzyme and substrate molecules which would result in a greater probability of successful collisions, and therefore more enzyme-substrate complexes being produced. This means that in my results, the volume of gas produced in the first 5 seconds may have been higher than it should have been if I had used exactly 0.2g of yeast. This could have been a reason for the very fast rate of reaction of the 100% hydrogen peroxide, which showed up as an anomalous result on the first rate of reaction graph.

    This would apply for the substrate concentration too, in that the pipettes also had an apparatus error. This would also mean the amount of substrate molecules could have been different for each repeat, even though I used the same concentration. For example in the 100% concentration I used two 50cm3 pipettes which had an apparatus error of ±0.01. So in 100cm3 the actual volume could have been either 99.98cm3 of hydrogen peroxide or 100.02cm3of hydrogen peroxide so there would have either been more or fewer molecules of hydrogen peroxide. If there were fewer molecules of hydrogen peroxide, there would have been fewer collisions between molecules of enzyme and substrate, resulting in fewer enzyme-substrate complexes being made.

    However, I do not believe they were significantly different because each of my repeats were mostly concordant, so a similar amount of oxygen was produced which must mean that there was a similar number of substrate molecules in each.

    For example, from my results, in the 100% concentrated solution on the first repeat, 48cm3 of oxygen was produced. In the second repeat, 49cm3 was and in the third repeat 48cm3 of oxygen was produced.

    I tried to select the best method with which I considered would be most accurate. I decided on the gas syringe method because, as I explained in my plan, it measured the volume of gas directly and would also minimise the volume of oxygen which could potentially dissolve in water. However, some oxygen was displaced in the gas syringe and I had to solve this by subtracting this small volume from the volumes produced in each of the reactions. Also, I noticed if the barrel was wet, the syringe often got stuck for a short time before it recorded the volumes of gas. To prevent this I had to dry out the barrel and syringe before commencing the procedure. It was very hard to insert the small 5cm3 beaker into the conical flask, and when it came to tipping it over, some of the substrate was still trapped inside the beaker. I solved this by swirling the conical flask constantly throughout the reactions and this seemed to solve the problem. Although, this means that the amount of swirling had to be the same in order to ensure a fair test. I tried to keep this constant by making sure I swirled the conical flask evenly. The accuracy of the results showed that this factor did not distort the results too much, and so a similar amount of substrate molecules were present in each reaction. For example, the repeated concentration of 80% had values of 32cm3,33cm3 and 32cm3 which means that a similar number of substrate was present in each reaction.

    Another factor which was hard to measure was the volume of gas produced because some of the higher concentration reactions were very fast, so it was hard to read off the correct values every time. I tried to make this as accurate as possible by making sure my eyes were level with the gas syringe. Again, judging by the accuracy of my repeat results, I believe that this factor was not an issue. Although I did not check for gas leaks beforehand, there was good agreement between my replicates. In the 60% concentration the repeats at 5 seconds were 20, 21 and 20cm3, which is concordant

    If my replicates had not been so close I would have had to change the tube.

    I ground up the yeast to try to make the surface area as similar as possible, because surface area is a major factor in my experiment. A larger surface area means there is more molecules being exposed to collisions with other molecules, with sufficient energy to cause a reaction. This means that having the same surface area of yeast in each reaction is very important in ensuring a fair test because the number of molecules exposed to collisions must be the same.

    Temperature is a major factor which affects rate of reaction. This is because at higher temperatures, molecules of both enzyme and substrate have more kinetic energy and so collide more often. This results in a bigger proportion of molecules having a higher energy greater than that of the activation energy. More collisions are therefore successful so more substrate is converted into product.

    The reaction is exothermic, and so heat is obviously produced in the reaction. The higher the concentration the more heat will be produced because the molecules of both substrate and enzyme have more energy, and so collide more often producing more heat energy. This heat energy is transferred to the environment.

    Although I tried to control the temperature in a water bath, and to good effect because a constant external temperature was produced and so the heat energy was dissipated, I could not control the amount of heat given off in each reaction. This would have affected my results for several reasons. Firstly, more oxygen dissolves in water at a lower temperature than at high temperatures and this means that for the reactions involving low concentrations more oxygen would have dissolved than in the higher concentrations because of the decreased amount of heat energy given off. Because the volume of oxygen dissolved in the reaction is not constant for all the reactions, because less oxygen is dissolved in water at higher temperatures, this would have affected my results. This may have been why the difference in the final volume of oxygen produced was not equal, but instead decreased in steps of 3.7cm3, 9.6cm3, 14.4cm3, 4.6cm3 and 7.7cm3.

    The different concentrations of hydrogen peroxide that I made up could not have been exactly accurate because this would have meant that the volume of gas evolved would have increased in equal steps which it did not. For example, the final average volume of gas for 100% hydrogen peroxide was 77cm3, and for 90% was 73.3cm3, 80% - 63.7cm3, 70% - 49.3cm3, 60% - 44.7cm3 and 50% it was 37cm3. As I have mentioned earlier, this decreases in steps of 3.7cm3, 9.6cm3, 14.4cm3, 4.6cm3 and 7.7cm3, which is far from equal.

    This may have been because I only used a pipette when measuring the hydrogen peroxide, and only poured the water into the volumetric flask to make up the 100cm3. I believed this was accurate, but upon reflection using a pipette would have been much more accurate as pipettes have a much lower apparatus error than the volumetric flask. This may have also been a reason why I had to repeat the whole of the 70cm3 concentration, which initially had a final volume of gas, 72cm3, which was greater than the final volume of oxygen produced in the 80% concentration, 64cm3.

    Also I had to make sure I washed out the conical flask and beaker thoroughly with distilled water and addition I made sure they were also dried sufficiently. If I hadn’t I could have risked diluting the solutions more and this would have affected the number of molecules of hydrogen peroxide present which in turn would have affected the number of collisions between enzyme and substrate molecules. For example, if there was still only 1cm3 of water still remaining in the conical flask and beaker combined, then in say the 80% concentration of hydrogen peroxide, the concentration would have been closer to 79%. This can be shown by the simple calculation of 80 ÷ 101 x 100 = 79.2%.

    Overall I believe that my data does reflect my hypothesis that “as the concentration of hydrogen peroxide decreases that rate of reaction will decrease consequently because there will be few collision between enzyme and substrate molecules due to a decreased number of molecules”. This is shown by my rate of reaction graph which shows that for the 100% concentration of hydrogen peroxide, the rate of reaction was 8cm3 second-1, and the 90% concentration was only 7.4cm3 second-1.

    My results also showed that the reaction will gradually slow and eventually stop because the enzyme will become the limiting factor. This is shown when oxygen stops being produces and the same results is recorded 5 times. So for example, I knew that the 100% concentration of hydrogen peroxide reaction was over because I recorded 88cm3 at least 5 times.

    However, I also believed that if I halved the concentration then the rate of reaction (volume of oxygen produced) would also be halved, and so the rate would be proportional to the concentration. This would show that the reaction is a first order reaction. Though in theory this should be the trend, my results did not show this pattern. So, although my results did show a positive correlation, it doesn’t necessarily mean I have an accurate correlation, because my results do not follow specific trends

    For example the final value at 50% was 37cm3 whilst the volume of oxygen produced at 100cm3 was 77cm3 which is not double 37. Again the final volume of oxygen produced at 30% was 27.3cm3, whilst the final value produced in the 60% concentration was 44.7cm3 which also not double.

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