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out how different concentrations of the enzyme pectinase affect the degradation of the substrate pectin

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PLANNING: - Aim: My aim is to find out how different concentrations of the enzyme pectinase affect the degradation of the substrate pectin, and hence the volume of clarified apple juice produced in a specific duration of time. Hypothesis: Will different enzyme concentrations have different effects on the yield of clarified apple juice obtained from apple pulp of equal masses, in a given time? Biological Knowledge: Enzymes are a class of proteins, which are biological catalysts responsible for speeding up and controlling metabolic reactions, without their shape being altered by the reaction. Pectinase (the enzyme I will be using for my investigation) is under the class of enzymes named hydrolases, which are responsible for catalyzing reactions between a substrate and water, and bind water to certain molecules. In this way, larger molecules are broken up into smaller units. This class of enzymes catalyses the cleavages of peptide bonds in proteins, glycosidic bonds in carbohydrates, and ester bonds in lipids. Enzymes are specifically designed for their target molecules, their substrates, by virtue of their shape, size, and the chemical charges that precisely fit and bond to the substrate molecule. The section of the enzyme that binds the substrate is called the active site. An advantage of using enzymes is that they are specific and do not interact with other components in the food or beverage. Another advantage is that by catalyzing the same reactions repeatedly as long as the substrate is available (referred to as turnover), they can be used at very low concentration. Eventually, as the end products of the enzyme reaction increase in concentration, the reaction is temporarily inhibited by feedback inhibition. This shows how the catalysis of pectin by the enzyme pectinase occurs: The cell wall of fruit comprises a complex mixture of various polysaccharides and some protein. Before fruit juice can be extracted from fruit, the cell wall has to be ruptured. ...read more.


Then I will take the 30g of apple pulp and place it in one beaker and with the same method I will weigh 30g of apple pieces and place them in the other beakers. Following that I will use a syringe to collect 4cm3 of distilled water and then squirt it over the apple pieces in the beaker labelled 'Control'. Using a different syringe I will suck up 2cm3 of the pectinase solution and 2cm3 of the distilled water and squirt it in the beaker labelled '50% Pectinase'. Then by the use of different syringes I will suck up 3cm3 of pectinase and 1cm3 of distilled water, and place the solution in the '75% Pectinase' beaker, and I will suck up 4cm3 of pectinase solution with no water and place the solution in the '100% Pectinase' beaker. Then I will stir the contents of each beaker with a separate glass rod. The four beakers will then be incubated in a water bath of 40?C for about 15 to 20 minutes. Meanwhile I will label four measuring cylinders, one 'Control', one '50% Pectinase', one '75% Pectinase' and the other '100% Pectinase'. I will place a filter funnel in each measuring cylinder and then I will place a filter paper in each of the filter funnels. After 15-20 minutes I will remove the beakers with the help of a small towel, and then take the contents of the beakers and place them into the filter funnels of the appropriate measuring cylinders, and start the stop clock. After ten minutes I will check the yield of apple juice produced for both experiments and record it. For my final experiment I will be finding the percentage change in comparison to my control (100% of water and 0% of pectinase). The formula which I will be using is the following: Average volume - average volume of the control X 100 Average volume of the control PILOT TEST: - I decided to have a pilot test in order to get ...read more.


This meant that the juice that had already come out of the apple pulp, before the pectinase was added, was not removed. This could have caused the volume of apple juice measured at each interval to be higher than would normally be expected and would reduce the reliability of my results. It was also difficult to have three experiments in progress at the same time, despite the fact that they were staggered by one minute, as two readings needed to be taken concurrently it still meant errors in reading could be made. This could mean that some experiments were left slightly longer than they should have been, resulting in an unfair test. Therefore the volume measured at each interval may not be very reliable. The fluctuations in temperature of the water bath may have affected the activity of the pectinase enzyme; more of the pectin molecules in the cell wall may have been broken down in one repeat than in another causing a higher average result than expected. As a result of all the above reasons my results may have varied a little between themselves, but this is why I repeated each concentration thrice and found its average volume, in order to avoid any variability in my results. I believe that my results are reliable because of this reason and also because my hypothesis was proved right and I received the expected results and expected graph trends. Improvements: I should be sure that I will measure the mass very carefully and with concentration. I have to be more precise when measuring the volume of pectinase solution and distilled with the syringe, taking care look at the lower meniscus; and doing the same when noting down the result from the measuring cylinder. I ought to rinse the chopped fruit with distilled water before the pectinase enzyme is added would definitely make the results more reliable. This would ensure that the juice produced was due to the action of the pectinase enzyme. Next time I could do each concentration at a time, instead of doing two simultaneously. ...read more.

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