An Investigation into the Effect of Lipase Concentration on the Hydrolysis Of Fats

An Investigation into the Effect of Lipase Concentration on the Hydrolysis Of Fats
Using the data loggers a recording of the pH was taken every 5 seconds and for each experiment the data loggers produced graphs of the change in pH. From each of these graphs a gradient was calculated which showed the rate of pH change per second. Firstly I calculated the gradients by choosing the steepest section of the graph and dividing the change in pH of this section by the time. However this method proved to be quite inaccurate giving very varied results, for example in these results the average rate of reaction for the 4% lipase solution (-0.457 pH/min) was lower than the 3% lipase solution (-0.471 pH/min). Also the rates in the 2% lipase solution ranged from -0.01 pH/min to -0.95 pH/min showing little reliability in the results. This was partly as I was only guessing which the steepest part of the graph was. I then used the analysis function within data loggers to calculate the gradient of the steepest part of the graph, this was a much more accurate method and the results are given below.
Results === Rate of Fat Hydrolysis (pH per Sec) % Concentration of Lipase Repeat 1 Repeat 2 Repeat 3 Average 1 -0.01 -0.01 -0.0100 2 -0.01 -0.01 -0.01 -0.0100 3 -0.02 -0.02 -0.01 -0.0167 4 -0.05 -0.06 -0.06 -0.0567 5 -0.03 -0.06 -0.06 -0.0600 Due to insufficient time a repeat for the 1% lipase could not be carried out, the first repeat for the 5% lipase (highlighted) looks to be anomalous so I have not included it when calculating the averages. I have plotted graphs from both sets of calculated gradients however I will concentrate on the graph plotted from the results show above as this shows a more definite trend and uses more consistent and reliable results. There is also a large amount of human error in calculating the gradients by without using the data loggers. Analysis The graph shows a definite decrease in the pH of the milk as the concentration of lipase enzyme increases. Two controls for these experiments were carried out, one with no lipase enzyme at all and one using a denatured version of the lipase enzyme. The graphs for these controls showed only slight fluctuations in the milk pH. The control with no lipase enzyme at all showed an increase of just 0.23 for the whole time period and the control using the denatured lipase enzyme showed a 0.06 decrease. These fluctuations could be due to slight inaccuracy in the data logger, they may also be due to the fact that the milk had slightly different pHs in different parts of the conical flask. The inaccuracy in the data logger may effect all the results for this experiment, which may have limited the accuracy of all the results obtained. As the experiments with the working lipase showed pH changes of around 3 this definitely proves that lipase enzyme lowers milk pH. [image] Hydrolysis -???- + 3 Water [image]Lipase enzyme breaks down fats into fatty acids and glycerol, as shown below; [image] Therefore when lipase enzyme is added to milk it lowers its pH as fatty acids are produced making the milk more acidic. The rate at which fatty acids are produced seems to increase as the concentration of lipase enzyme increases. This is due to an increased number of collisions between the lipase enzyme active sites and the fats in the milk. The lipase enzyme lowers the activation energy required to start the breakdown of fats allowing the reaction to take place at a lower temperature. Therefore the more lipase enzyme present the more the rate of reaction is increased. Only the lipase enzyme will lower the activation energy of this specific reaction, this is due to the tertiary structure of the enzyme, which determines the shape of the lipase enzyme?s active site. The active site is where the fat substrate binds so the lipase enzyme?s active site must be complementary to the shape of the fat substrate. The fat substrate binds onto the active site of the lipase enzyme forming an enzyme-substrate complex. The reaction takes place and the products, fatty acids and glycerol are released, and the lipase enzyme remains unchanged. With an increased percentage of lipase enzyme in the same volume there is more likely to be collisions between active sites and fat substrate. This results in an increased number of collisions per second and therefore a quicker breakdown of fats into fatty acids and glycerol. Hence a higher concentration of lipase enzyme causes a faster fall in milk pH. However there is still roughly the same overall pH fall in all the experiments of different lipase concentrations. This is because although there are more collisions per second for higher lipase concentrations, there is still only the same amount of fat substrate so only the same amount of fatty acids being produced. [image] The above graph shows how the rate of fat hydrolysis increases with lipase concentration, however the rate only increases up to a certain point. At the point where the graph levels out other factors are limiting the rate of fat hydrolysis in this case the main limiting factor is the amount of fat substrate. The graph did not seem to show a direct straight-line relationship between increase in lipase concentration and increase in rate of lipid hydrolysis. I calculated the correlation of the data using the product moment correlation coefficient formula and found the correlation coefficient to be 0.91. Therefore the data follows a straight-line format quite closely and the slight curve shown by the trend line could be put down to anomalies in the data. However it may be caused by the faster change in pH for more concentrated lipase concentrations. The lipase enzyme works better at the lower pH as it is be nearer to the enzymes optimum pH which ranges from about 4-8. A change in pH alters ionic charges of acidic and base groups, the tertiary structure of the enzyme is altered changing the shape of the active site so the substrate cannot bind. Therefore the closer the pH is the lipase enzyme?s optimum pH the faster the rate will be. This would explain why the rate for higher concentrations is so much higher than that of the lower lipase concentrations. From the data it can be seen that the higher the concentration of lipase enzyme the faster the rate of fat hydrolysis into fatty acids. Evaluation The data loggers were used as they were a much more accurate method of measuring the pH than using universal indicator. As using the data loggers it was possible to take readings every few seconds this would not have been possible using universal indicator. Also using universal indicator there would have large amount of human error as it would have only be a rough guess what the pH was using the colour whereas the data logger is able to record pH to two decimal places. However the pH probe could not detect the pH throughout the milk and so stirring the milk around was necessary. This may have caused inaccurate pH readings as although the milk was mixed it was not mixed constantly and certain areas of the conical flask may have been more deficient in fatty acids giving a higher pH reading than was accurate. Another cause for anomalous results may have been the measuring of the milk and lipase. The milk was measured using measuring cylinders, a more accurate method would have been to use graduated pipettes. Also the lipase enzyme was measured using measuring cylinders and then diluted to the correct concentration using water. Even slight variations in the concentrations of the lipase solutions could results in large variations in the rate of lipid hydrolysis. The lipase solution was made from a powder and although before using the enzyme the solution was mixed, it could still have been varying concentrations throughout the solution, this could again cause quite large differences in the rate of lipid breakdown. The temperatures at which the experiments were carried out would have affected the rate at which the lipase enzyme worked. Although the experiments were not tested before to ensure all temperatures were the same, the milk and lipase came from the same storage place in each case. This would have minimised temperature variation between the experiments. However all the experiments were carried out in different parts of the lab, drafts from doors and windows may have meant slightly lower temperatures for some experiments and may have caused the lipase enzyme to work more slower as it may have been further from its optimum temperature. The increased temperature in different parts of the lab would have caused more collisions between lipase enzyme and fat substrate, as the particles would have had more kinetic energy, this would have increased the rate o reaction. The rate of reaction would have increased up to the lipase enzyme?s optimum temperature here hydrogen and ionic bonds would break changing the enzymes tertiary structure so the fat substrate would no longer fit- the enzyme would have become denatured. Once the anomalous result was removed from the calculations the repeats for each lipase concentration seemed to be consistent. For the 2% concentration of lipase all three repeats came out with a gradient of 0.1 and all the other repeats were with 0.1 of each other. At least two results were used for calculating each average gradient and all repeats were similar, the results gained are reliable enough to support the conclusions made in the analysis. The one anomalous result could have been down to any of the factors mentioned above. Using the data loggers pH change could only be calculated in pH change per second a more accurate gradient would have been in pH change per minute. The pH only changed very small amounts (hundredths of a pH) in a second whereas it can be seen from the gradients calculated by hand that the pH change in a minute was much larger as least 0.3 so slight inaccuracies would have not been as important. Also the data loggers would only give the gradient to 2 decimal places meaning many of the gradients were the same. Despite these inaccuracies in data analysis I feel the results are reliable enough to support the conclusions made.

An Investigation into the Effect of Lipase Concentration on the Hydrolysis Of Fats 7.8 of 10 on the basis of 4051 Review.