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To carry out an investigation into the denaturing of immobilised lactase enzymes in relation to thermodynamics.

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Planning Aim To carry out an investigation into the denaturing of immobilised lactase enzymes in relation to thermodynamics. Introduction Lactose is a disaccharide and is the sugar naturally present in milk. During the manufacture of milk products, the water contained in milk must be removed in order to thicken the product. This concentration can be done in various ways however always results in a significant loss in lactose. Due to this, those with some degree of lactose intolerance more easily consume products. Although milk products are highly regarded by consumers for their nutritional value and therefore easy to sell, lactose presents hardly any commercial value. Food manufacturers often don't know what to do with it and often end up disposing of this sugar in effluents to the rivers. One of the reasons that make it difficult to find a use for lactose is the fact that some consumers are unable to digest it. This makes the use of lactose a highly important issue in many parts of the world. Currently, lactose is used primarily as a filler or sweetener, and can be found in small amounts in infant and dietary foods and coffee whiteners. Lactose is very inexpensive due to its existence in great amounts. The splitting of lactose can be brought about with the use of the enzyme, lactase, into its components, the monosaccharides glucose and galactose. These can then be put to use as sugars. (Lactase) Lactose Glucose + Galactose (disaccharide) (monosaccharide) (monosaccharide) Lactase, a biological catalyst (enzyme) can be found in the body, and is specific to the digestion of lactose. Lactase works best in temperatures of between 40?C-45?C. I am going to investigate whether the same is true for lactase when it is immobilised, and how temperature affects its catalytic activity. Hypothesis Enzymes act as biological catalysts; they increase the rate of chemical reactions. The substrate is the substance with which the enzyme combines; it does so at a particular place on the enzymes surface referred to as the active site. ...read more.


No results in this experiment could be used as part of this investigation other than for the purpose of learning from mistakes. Evaluation The enzymes used in this experiment were slightly soft. To prevent this in the next experiment the enzyme solution must be mixed thoroughly before being added to the calcium chloride solution for immobilisation. The accuracy and fairness of the experiment could be improved by wrapping bubble wrap around the beaker once the milk reaches the required temperature and the bunsen burner has been turned off. This insulation will help maintain the milks temperature for a longer period of time before cooling down, ensuring it does not decrease whilst the experiment is still taking place. The fairness of the experiment could also be improved by ensuring immobilised beads are roughly the same size by measuring the drops e.g. 2ml of enzyme solution added to the calcium chloride solution. This will help keep the surface area of the enzyme variable constant for a fairer test. As the Benedict's test did not work no quantative results were gained. In an experiment such as this I believe the most quantative results possible will only be gained with the use of glucose strips. In my final experiment I will record the number of crosses (which relate to the amount of glucose present), in accordance with the glucose strip scale, per sample and shall test at 5 samples per temperature at 1-minute intervals up to 5 minutes. I shall plot a graph of results and observe any trends. Due to the expense of the Diabur 5000 Glucose Strips and as I am taking results at greater intervals I shall reduce the number of temperatures investigated to 40?C, 60?C, and 80?C. I shall also repeat the experiment at least three times and calculate an average from the range of results taken to ensure that anomalous results do not affect my conclusion. ...read more.


The Benedict's test would be negative due to the non-reducing sugar, so then the sample would be hydrolysed by heating with hydrochloric acid, then neutralised with sodium hydrogen carbonate. This breaks the glycosidic bonds present. Then a second Benedict's test would be performed, which will be positive because the monomers are now free. Despite these improvements, I believe I carried out this investigation to the best of my ability especially considering the limitations. The limitations I faced included limited time, the expense of the Diabur 5000 glucose strips, and the amount of lactase enzyme available. All of these confined my investigation in some way resulting in the investigation not being carried out to its full potential. In spite of this the results that I was able to gain were very successful. Results Graphs The following are small and simple graphs to show the general trend in how temperature affects the amount of glucose present through a range of 1-minute to 5-minute reactions: Results after 1-minute time interval Results after 2-minute time interval Results after 3-minute time interval Results after 4-minute time interval Results after 5-minute time interval The following is a more complex graph displaying all the results obtained from the investigation. The graph shows the effect of temperature on the amount of glucose produced within 1-minute time intervals for 5-minutes. Results Time intervals after which samples were taken (after contact between milk and enzyme) 1st experiment 2nd experiment 3rd experiment Temperature 1min 2mins 3mins 4mins 5mins 1min 2mins 3mins 4mins 5mins 1min 2mins 3mins 4mins 5mins 40?C Neg. + ++ +++ +++ + + ++ +++ +++ Neg. + ++ +++ +++ 60?C + ++ +++ +++ +++ + + +++ +++ +++ + ++ +++ +++ +++ 80?C Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. +++ +++ +++ +++ +++ (Neg. = Negative result [no change on glucose strip]) Average Results Time Intervals Temperature 1min 2mins 3mins 4mins 5mins 40?C Neg. + ++ +++ +++ 60?C + ++ +++ +++ +++ 80?C Neg. Neg. Neg. Neg. Neg. ...read more.

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