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Investigate the effect of enzyme temperature on the activity of the enzyme Trypsin on the substrate Casein.

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Introduction

The Activity of the Enzyme Trypsin on the Insoluble Milk Protein Casein Planning (skills A and B): - Aim: - To investigate the effect of enzyme temperature on the activity of the enzyme Trypsin on the substrate Casein. Introduction: - Casein is an insoluble protein found in powdered milk, such as Marvel, which forms a white or cloudy suspension (not dissolved). A white suspension of Marvel milk in water clears if a protein-digesting enzyme is added. The enzyme used in this experiment is trypsin. When the suspension is hydrolysed (broken down), it starts off cloudy, but becomes clearer as the products (peptides and amino acids) dissolve. This reaction is catalysed by proteolytic enzymes (proteinases) such as trypsin. Trypsin is released into the small intestine in alkaline conditions. It is one of the main digestive proteinases, as well as pepsin and chymotrypsin. Trypsin acts with other proteinases to break down proteins to peptides and amino acids. It just continues the digestion process that was begun in the stomach in acidic conditions. Trypsin is produced in an inactive form by the pancreas. Its chemical composition and structure is extremely similar to chymotrypsin. Both enzymes appear to have similar mechanisms of action as they have residues of histidine and serine in their active sites. The difference between them is their specificity. Each is active only against the peptide bonds in protein molecules that have carboxyl groups donated by certain amino acids. For trypsin these amino acids are arginine and lysine. Trypsin is the most discriminating of all the proteolytic enzymes in terms of the restricted number of chemical bonds that it will attack. This means it is widely used to determine the sequence of amino acids in proteins and as a reagent. Trypsin is an enzyme that acts to degrade proteins. It breaks down the casein into polypeptides which are soluble. That's why the solution goes clear. ...read more.

Middle

* Record how long it took for all three test tubes to clear, and then wash them out using distilled water. * Draw up 15ml of trypsin using the same syringe. It is extremely important the casein syringe is not used as it will lead to inaccuracies in the experiment. 15ml is drawn up instead of 5ml because it is more efficient, as there are 3 test tubes needing 5ml of trypsin each. * Place 5ml of trypsin in each of the test tubes. Place a thermometer in all three test tubes and place them in the water bath set to 30oC. The trypsin is placed in the water bath, not casein, because I am investigating enzyme temperature. The casein would also get denatured at high temperatures, as well as trypsin, because it is a protein. * Get the stopwatches ready, and draw up 15ml of casein using the right syringe. * Wait for the temperature to reach 30oC on the thermometer then immediately insert 5ml of casein to the test tube and start the stopwatch. * Stir the solution using the thermometer from time to time. * Do the same for the remaining two test tubes and use the distilled water again as a reference as to when to stop the stopwatch. * Record the time taken and wash out the test tubes using distilled water. * Repeat the procedure at temperatures of 40oC, 50oC, 60oC, 70oC and 80oC. * The test tubes are put in the water bath three at a time at the same temperature because this repeats the results in exactly the same conditions. Therefore, my results will be more reliable and accurate. Safety: - * Keep the experiment away from the edge of the table to avoid spillage. * Wash your hands if any enzyme gets onto it immediately. * Tie back any loose objects (i.e. hair) as they may interfere with the experiment and loose objects can hook onto apparatus and tip it over. ...read more.

Conclusion

This was quite effective as I had done a pilot experiment to see how clear the mixture goes, and it was roughly the colour of the distilled water bottle. The same syringes were used for putting in the casein and trypsin, and this ensured there was no previous contamination, and made my experiment more accurate. If I had used a new syringe, it may have had remains of other chemical substances that would have affected my experiment. I tried to keep the conditions the same as I used the same test tubes, the same syringes, the same thermometers, the same stopwatches, the same batch of casein and trypsin, and the same water baths. My experiment was quite reliable as I repeated the results three times, and I even re-investigated the anomalous results. I think the procedure was fairly accurate as I used mainly measuring devices containing fixed error, which was cancelled out. The main problem was human error. The only way to improve the procedure is to use specialist equipment to replace the job given to us, and therefore minimising errors. The number of experiments is obviously a limitation. If I had the opportunity, I would test more temperatures, and repeat the results more times to make the procedure more accurate and reliable. I obtained two anomalous results due to my inaccurate procedure and they were 40oC and 50oC. They came about due to the stirring regime, which was to use the thermometer as a stirring rod from time to time if the mixture wasn't clearing fast enough. This was obviously not a good idea for the optimum temperatures as they clear in a matter of seconds and you may not get time to stir it properly, or you may stir one test tube more vigorously than the other. A magnetic stirrer would make the procedure much more accurate. Other than that I think my procedure was quite good as I obtained quite good results and I followed the right safety precautions, ensuring my procedure was safe, efficient, accurate and reliable. Aisha Hussain Page 1 08/05/2007 ...read more.

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