To find out what factors affect the rate of reaction between rennin and milk.
AimTo find out what factors affect the rate of reaction between rennin and milk. SECTIONS OF THIS REPORT Research Enzymes Enzyme Action Specificity Reversibility Temperature pH Other Factors which could Affect the Rate of an Enzyme Rennin Predictions Temperature pH Concentration of Enzyme Which Variable has the Greatest Relative Effect? Planning Measuring the Rate of Reaction Quantities Plan for Temperature Plan for pH Plan for Concentration of Enzyme Accuracy Results Temperature pH Concentration of Enzyme Conclusions How Temperature Affects the Rate of Rennin How pH Affects the Rate of Rennin How the Concentration of Enzyme Affects the Rate of Rennin Overall Conclusion RESEARCHI know that Rennin is an enzyme, so before conducting this experiment I am first going to do some research into enzymes and their effects. 1. EnzymesEnzymes are large globular molecules of which the vast majority are protein in nature, though some, known as 'ribozymes' are made of RNA. Enzymes have catalytic properties; in other words, they alter the rate of reaction without themselves undergoing a permanent change. Most chemical reactions require an initial input of energy, called activation energy, to enable them to occur. Enzymes reduce the need for activation energy and so allow reactions to take place more readily and at lower temperatures than would otherwise be necessary. This can be seen in the graphs. 2. Enzyme ActionEnzymes, as biological catalysts, can be used in both anabolism (the build up of simple chemicals into complex ones) and catabolism (the breakdown of complex chemicals into simpler ones), although the latter is more common especially in the animal digestive system. As shown in the next diagram it is thought that the substrate molecules fit precisely into the enzyme molecules. This theory is referred to as the lock and key mechanism. However, in practice, it is likely that the enzyme itself to some extent is the substrate. The part of the enzyme molecule into which the substrate fits is called the active site. The configuration of the enzyme is due to ionic bonding, hydrogen bonding, disulphide bridges and hydrophobic interactions. 3. SpecificityThe substrate molecule makes a precise fit into the active site and though the enzyme may be flexible up to a point, the number of molecules which can fit into the active site is very small - in fact it is often limited to just one type. Therefore enzymes are specific to one type of reaction. 4. ReversibilityEnzymes can catalyse the forward and reverse reactions equally. 5. TemperatureAs temperature increases, the molecules, according to the 'Kinetic Theory' move faster, due to increased energy. Therefore, the enzyme and substrate molecules will meet more often and the rate at which the product is formed will increase. However, as the temperature continues to rise the hydrogen and ionic bonds, which hold the enzyme in shape, break and the active site will no longer accommodate the substrate. The enzyme is then said to be denatured - this cannot be reversed. 6. pHEfficient functioning of an enzyme depends upon the shape of its active site. This shape is determined, in part, by ionic and hydrogen bonding - which can be affected by pH. Therefore every enzyme has its optimum pH, when its substrate fits exactly into its active site. Variation of pH will denature the enzyme. 7. Other Factors which could Affect the Rate of an EnzymeInhibitors compete with the substrate for the active sites of enzymes. They are known as competitive inhibitors. The greater the concentration of the substrate the more likely it is to occupy the active sites and the less the effect of the inhibitor. Non-competitive inhibitors attach themselves to the enzyme at a site other than the active site. However, in doing so they alter the shape of the active site in such a way that the substrate cannot fit into it and the enzyme cannot function. As the substrate and inhibitor are not competing for the same site, an increase in substrate concentration does not diminish the effect of the inhibitor. 8. RenninRennin is a proteolytic enzyme and is characteristically found in the gastric juices of young mammals. It is one of only two enzymes (the other being pepsin) to be produced in the stomach. Rennin is secreted in an inactive form, pro-rennin, which is activated by the hydrochloric acid of the gastric juice. Rennin catalyses the conversion of the protein of milk, caseinogen, into paracasein, which is precipitated in the stomach as a calcium salt. The precipitated paracasein forms a firm curd in the stomach, which ensures that milk stays for some time in the stomach so that it becomes exposed to the actions of the proteolytic enzymes and the acid in the gastric juice. REFERENCESFor my research, I used 'Letts Study Guide' by Glenn and Susan Toole and 'Biology of the Mammal' by P. Catherine and Arthur G. Clegg. MY INVESTIGATIONThe factors that affect the enzyme rennin which I am going to investigate are: Temperature pH Concentration of enzyme PREDICTIONS9. TemperatureHow will temperature affect the rate at
which rennin acts on milk? The Kinetic Theory states that with increased temperature, molecules receive more energy, resulting in them speeding up their movement. If the milk and the rennin molecules are moving faster, they will collide more often. The collisions will also have more energy so more of them are successful. Therefore the rate of reaction increases. However, above 37°C, which I believe is rennin's optimum working temperature, because this is body temperature, and rennin is found within the stomach of young mammals; I predict that the rate of reaction will decrease. This is because the rising temperature affects ...
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which rennin acts on milk? The Kinetic Theory states that with increased temperature, molecules receive more energy, resulting in them speeding up their movement. If the milk and the rennin molecules are moving faster, they will collide more often. The collisions will also have more energy so more of them are successful. Therefore the rate of reaction increases. However, above 37°C, which I believe is rennin's optimum working temperature, because this is body temperature, and rennin is found within the stomach of young mammals; I predict that the rate of reaction will decrease. This is because the rising temperature affects the hydrogen and ionic bonds which determine the shape of the enzyme. As these bonds are broken the shape of the active site changes and the molecules of caseinogen can no longer occupy them. The rennin is now denatured and the rate of reaction will become zero. This is irreversible. If the temperature falls below 37°C, according to the Kinetic Theory the enzyme and substrate molecules will not be receiving as much energy, and therefore their movement will slow down. This will reduce the number of collisions between the rennin and the caseinogen, and there is a higher probability that these collisions will be unsuccessful due to the lack of energy. Therefore the rate of reaction will reduce. At 0°C I still suspect the rennin to catalyse the conversion of caseinogen to paracasein, but it will take a lot longer. I predict that to stop the reaction altogether it would have to be at absolute zero (-273°C) when Scientists predict that there would not be enough energy for the movement of any molecule at all. The graph shows how my predicton of how temperature will affect the rate of rennin. The actual rate of reaction that will take place will be a balance between the two opposing influences of greater kinetic movement of molecules increasing the rate and the decreasing rate due to denaturation of the rennin. 10. pHHow will the pH affect the rate at which rennin acts on milk? I know from my research that rennin is produced in the stomach and is activated by the gastric juice. The gastric juice contains much hydrochloric acid, and therefore I predict that rennin will work to its optimum around pH 1. Variation from this pH, I believe will slow the rate of reaction because the differing pH will affect the ionic and hydrogen bonding which determine the shape of the enzyme. When the active site of rennin is affected, the molecules of caseinogen will not be able to fit precisely into the site and therefore less or no product will be produced, and therefore the rate of reaction will decrease. I predict that rennin will still work in neutral conditions because milk is neutral and therefore rennin cannot be denatured by neutral conditions. Also the hydrochloric acid in the stomach is diluted by the other gastric juices, food and milk, so the rennin must be able to work in weak acids. However in extreme alkali conditions I predict the rate of reaction to be zero because varying the pH can change the bonds within the enzyme, and therefore change the shape of the active site until the substrate is unable to fit into it - ie it is denatured. 11. Concentration of EnzymeHow will the concentration of the rennin affect the rate of reaction? I know enzymes function efficiently in low concentrations as the molecules can be used over and over again. However, I predict that an increase in the concentration of rennin will lead to a corresponding increase in the rate of reaction up to a point. For when there is enough enzyme to hold every molecule of substrate, an increase in enzyme concentration will have no effect. The graph shows how the rate of reaction will be directly proportional to concentration up to the limiting point. 12. Which Variable has the Greatest Relative Effect?I think that temperature will have the greatest effect on the rate of reaction. For I have predicted that relatively small changes in temperature produce a large change in the rate of reaction of rennin. For without enough energy provided by heat rennin cannot have as many successful collisions with caseinogen, and the reaction is very much hindered. Temperature also can completely stop the rennin by denaturation. The other two variables, I predict, will have a less overall effect on the reaction. For I know enzymes work very economically in small concentrations because they can be used over again, and therefore variation in concentration will have no dramatic effect on the rate of reaction. I know that rennin works best at pH 1, however, I predict that it will continue to break down caseinogen in pH higher than this because in the stomach of a mammal, where rennin is found, the hydrochloric acid is diluted by the other liquids in the stomach and the food or milk that the animal has eaten. Therefore, rennin must be able to work in acid much weaker than pH1 which suggests that pH will have less overall effect. PLANNINGTo get successful and accurate results I am going to test each of the variables (temperature, pH, and concentration) separately whilst ensuring that the remaining two remain constant. 13. Measuring the Rate of ReactionRennin, I know, catalyses the conversion of the protein of milk, caseinogen, into paracasein, which precipitates as a calcium salt. Therefore by timing how long it takes this to occur, ie for milk to clot, I will be able to get an indication of the differing rates of reaction. To ensure a fair test, I am going to stop the stop-clock when the milk has fully clotted and the whey is fully separated from the curd. The faster it takes to clot, the faster the rate of reaction. 14. QuantitiesFor each experiment, so that I can compare them in my conclusion, I am going to use 2ml of milk and 4 drops of rennin (obviously for the concentration experiment, this will vary). To ensure a fair test each experiment will be conducted with the same type of milk - semi-skimmed. 15. Plan for TemperatureTo study how temperature affects the reaction between milk and rennin, I am going to look at a range of different temperatures between 0°C and 80°C. I chose a minimum temperature of 0°C because it is the coldest temperature I can achieve with the apparatus available to me. I chose a maximum temperature of 80°C because at this temperature I would expect, after my research and prediction, for all the rennin to be denatured. Also 80°C is still a safe temperature to work at, and the milk won't curdle too quickly. I am going to use temperatures at 20°C intervals within the 0°C to 80°C guidelines so that I am able to get detailed results whilst ensuring that there is a significant difference in the rate of reaction. To keep all other variables the same I am going to use the same amount of milk (2ml) each time and use the maximum concentration of rennin. I know milk to be of neutral pH after testing it with universal indicator paper and have decided to leave it so. For, though I know rennin to react best in acidic conditions, by adding hydrochloric acid to provide favourable conditions, I would be unsure whether it was the acid or the temperature having the most effect on the reaction, and I would have inaccurate results. Also, hydrochloric acid separates milk itself, so the reaction would begin before the rennin was added. When heating or cooling, I will do exactly the same to both the milk and the rennin, to ensure that both are at the same temperature and it is not just the milk being heated while the rennin remains at room temperature, which would, of course, effect the results I received. For if the milk was at 80°C, but I had not heated the rennin, the enzyme would still be able to react with the milk until eventually the rennin itself had reached a high temperature and was denatured. To ensure that the enzyme and substrate remain at the required temperature I am going to pour them together and watch for the reaction whilst they are still in their set conditions, ie I will not take the boiling tube out of the water bath. I am going to use ice and water baths, heated or cooled to different temperatures to achieve the correct temperature in the enzyme and substrate. 16. Plan for pHI am going to use the three categories of acid, neutral and alkaline to test how pH affects the rate of the enzyme rennin. Whilst studying the affects of varying the pH, I am going to ensure that all other variables are constant. Therefore, I will continue to use 2ml of milk and 4 drops of rennin. The temperature I shall conduct these experiments at will be room temperature (27°C). I have decided on this temperature because it is hot enough to provide the enzyme and substrate with enough energy for a successful collision and yet it is not quite ideal. For if it was the ideal temperature to have the rennin, the reaction would take place so fast that no significant difference between the various pH levels would be seen; for I have predicted temperature to have the greatest relative effect. To change the pH, I am going to put 2 drops of the following into the rennin: hydrochloric acid for acidic conditions (pH1) water for neutral conditions (pH7) sodium hydroxide for alkaline conditions (pH10) I will mix the two together and leave for one minute to allow the pH to have an effect on the environment of the rennin. I am not going to put the acid, water or hydroxide into the milk because I know that the hydrochloric acid will begin the conversion of the caseinogen to paracasein before the rennin has been added. Also the milk will dilute the pH. To see if this happens with any other pH, I am going to set up a control for each experiment where no rennin is added, but the pH of the milk is changed. I must ensure in this experiment that none of the substances mix through pipettes or thermometers otherwise my results will be inaccurate. I will then time, as usual, the length of time it takes for the milk to clot. 17. Plan for Concentration of EnzymeTo investigate how the concentration of enzyme affects the rate at which it works I am going to vary the number of drops of rennin I add to the milk. However, to keep the volume of the liquid the same I will make up the rest with drops of water. number of drops --------------- rennin water ------ ----- 4 0 100% enzyme 3 1 75% enzyme 2 2 50% enzyme 1 3 25% enzyme 0 4 0% enzymeI will again use 2ml of milk; have everything at a temperature of 27°C; leave the milk and rennin in neutral conditions. This is so that the reaction is not completed so fast that I cannot measure it, and also to ensure the effects are purely due to the concentration of the rennin. 18. AccuracyTo ensure that my results are accurate I am going to conduct each of these experiments twice. RESULTS19. TemperatureThe table shows the results I collected when I altered the temperature. I was only able to get results from this experiment at two temperatures (20°C and 40°C). Above these no reaction occurred at all which suggests that the heat had denatured the rennin. Below 20°C at freezing point I was forced to stop timing due to the limitations of time. The milk had begun to clot but had still not finished after 50 minutes. 20. pHThe table shows the results I collected when I altered the pH. 21. Concentration of EnzymeThe table shows the results I collected when I altered the enzyme concentration. CONCLUSIONS22. How Temperature Affects the Rate of RenninThe fact that I only achieved results at two temperatures shows that rennin is greatly affected by variations in temperature. here is also a vast difference between the time of reacting for 40°C and 20°C - 21.95 minutes in fact. This shows that, as I predicted, rennin's optimum temperature is round about body temperature. This is the temperature when the conflicting influences of increased rate of reaction due to increased heat energy and the decreased rate of reaction due to the denaturation of the rennin is most balanced. For at 40°C, the rennin has sufficient energy, due to the heat, to have increased kinetic energy as do the milk molecules. Thus with the faster movement more collisions will take place, and as the enzyme and substrate have high energy there is a greater chance of there being enough energy to reach the activation energy and for the conversion to take place. However at 40°C the enzyme is not (or is only mildly) affected by the destruction of its hydrogen and ionic bonds by the heat. When they are broken the shape of the active site is changed and the caseinogen will no longer fit into the site and the reaction stops, therefore the rennin is said to be denatured. I believe this to have occurred at the temperatures 60°C and 80°C. The measurement for 20°C is so much longer than that for the optimum temperature of 40°C because again of the kinetic and collision theories. For the reduced heat reduces the amount of energy the molecules are receiving. This decreases their kinetic energy, ie their movement slows, which in turn decreases the amount of collisions between enzyme and substrate, and increases the likelihood that there will not be enough energy for the reaction to take place when the rennin and caseinogen collide. Therefore the rate of reaction reduces. I expect that at 0°C the rennin and caseinogen had such a lack of energy that it would have taken them a long time to react together. I stopped timing at 50 minutes, but I believe the milk would have eventually fully clotted for I could see that it had already begun to do so. My results are as I predicted with rennin being denatured at high temperatures and being unable to react at low ones. However, I did not realise how small the range of temperature was that it works in. For reacting milk and rennin at temperatures other than about 40°C is very uneconomic due to the time it takes to clot. So 40°C appears not only to be the optimum temperature at which the rennin works but nearly the only temperature at which it achieves the conversion of caseinogen to paracasein. It is due to this that I now realise how important it is for babies to be fed milk that has been gently heated so that the rennin in their stomachs will work successfully. If I was able to do this experiment again I would like to make the range of temperatures even smaller, especially by breaking down the range 20°C to 40°C into maybe 5°C intervals. This would enable me to achieve an even more specific and accurate temperature at which rennin works to its optimum. The table shows that the results for the two times I did the experiments were very similar and so I can say that my experiment was fairly accurate, within experimental limitations. 23. How pH Affects the Rate of RenninI can see from my results that the rennin is completely denatured by alkali conditions, as predicted. For this pH has altered the bonds in the enzyme which help the rennin keep its shape. Thus when these bonds are altered the shape of the active site of the rennin changes which prevents the substance fitting into it, which prevents the reaction taking place. I can see that the rennin does work in neutral conditions, but not as quickly as when it is in acidic ones. This is as predicted because the gastric juice in the stomach contains a lot of hydrochloric acid and this is where rennin is produced. I believe that rennin is not denatured by neutral conditions, because rennin has to be able to work when the acid in the stomach is diluted by food and other liquids. Also milk is of a neutral pH, so if rennin was denatured by such, problems would occur. Also neutral is not an extreme pH, as alkali is, and so is closer to an acidic pH. The control shows that hydrochloric acid breaks the milk down, unlike water or sodium hydroxide, without the need for rennin. However clotting does not occur as quickly without the catalyst. This was to be expected because as mammals mature the production of rennin is reduced and it is left primarily to the acidic gastric juice to convert the milk protein. This experiment proves that the rate of rennin is affected by varying the pH, and that its optimum working pH is acid, pH1. If I had had more time I would have liked to dilute the acid to achieve a range of pH between 1 and 7. this would enable me to see how smaller changes in pH affect the rate of reaction. The problem with this experiment is that though I know the rate of reaction to be best at pH 1, from the control I also know that acid itself breaks up the milk. Therefore, how much my result is the increased rate of the rennin due to the acid or how much is due to the acid itself converting the caseinogen to paracasein I cannot be certain. However, it took a much longer time for the acid to clot the milk without the rennin, which shows that when it was added the rennin was a lot more active and successfully catalysed the reaction. From my table I can see that the results of the initial and repeat experiments were very similar, suggesting that both were fairly accurate. 24. How the Concentration of Enzyme Affects the Rate of RenninI can see that as predicted the highest concentration of rennin produced a clotting in the shortest amount of time and therefore had the highest rate of reaction. My results show no sign of the presence of the limiting factor, meaning that there was still enough substrate for the enzyme to act on in the maximum concentration. In a further experiment I would like to increase the ratio of rennin to milk until I can tell that all substrate is occupying an enzyme molecule, and the addition of more rennin would have no effect. As the concentration decreases so does the rate of reaction, as I predicted, for it takes more time for the fewer numbers of rennin molecules to convert all the milk molecules. As in my prediction the concentration of the rennin will not completely stop the experiment (unless it is not there as in 0% conc.) because rennin is not affected by the reaction it catalyses, so it can be used over and over again. However, in the smallest concentration I was forced to stop timing because the reaction was taking so long. I feel it is important to note that if the rennin had been heated to its optimum temperature of 37°C to 40°C, even in that small concentration it would have had the ability to move faster and collide more successfully and therefore would have clotted the milk at a faster rate. A limitation with this particular experiment is that the volume of liquid in each pipette drop can vary slightly, so the results may not be truly accurate. 25. Overall ConclusionFrom the results and conclusions of the three independent experiments, I am able to state that the ideal working conditions for rennin is in a temperature of 40°C, in a pH of 1 and in a maximum concentration of enzyme. I believe that my predictions were correct and temperature does have the greatest relative effect; for the concentration of rennin will not completely stop the reaction or dramatically increase it, because enzymes can be used over again. pH is a very important factor because in alkaline conditions the enzyme is denatured and the difference in reaction times between neutral and acidic conditions are quite great. However, by looking at the results for temperature I can see that it causes the reaction to stop above 40°C because the enzyme is denatured and below 20°C it has the effect of slowing the reaction dramatically and so I was unable to measure it. The considerable difference between the times for 20°C and 40°C suggests to me that just small variations in temperature have great affects on the rate of reaction. The primary limitation with this experiment was the judgement by eye of when the milk had fully clotted, as this is not going to be completely accurate and the same for each experiment. I considered conducting the experiment in another way, but as no gas, which I could collect for example, was produced, the only other way was to weigh the amount of curds and whey created. However, with thought I reasoned that the factors I was testing would not affect the actual amount of curd produced, just the speed at which it was produced. If I was to do this investigation again I would use more detailed and smaller ranges of readings because for many of the experiments I got no result, as it appears rennin is very specific in its ideal conditions. To conclude I have found rennin to be denatured in alkali conditions and/or temperatures over 60°C, but as predicted, to work best at 40°C, at pH 1 and with the maximum amount of the enzyme possible.
