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Investigation into comparing free to Immobilised amylase enzyme in its catalysis rate

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Investigation into comparing free to Immobilised amylase enzyme in its catalysis rate Method: First of all, the Immobilised enzymes need to be made. The method used to create these immobilised enzymes would be Micro encapsulation. This means that the enzyme used, in this experiment being Amylase, is encapsulated inside Sodium alginate. The enzyme was believed to act quickly, so the enzyme would have to be slightly diluted in order to get a good range of results. !0cm� of Amylase will be added to 20cm� of Sodium alginate, and then mixed. Using a pipette, the Sodium Alginate - Amylase mix is dropped into Calcium Chloride, and this will form the beads of Immobilised enzymes needed for the experiment. As soon as Sodium Alginate touches Calcium Chloride, it solidifies and this traps the enzyme inside. The good thing is that the beads are semi permeable, so the substrate can penetrate the bead and get to the enzyme. As I'm sure you are well aware, enzymes do not get used up in a catalysis reaction, so the beads can be used again and again to our benefits. The Immobilised enzymes will be kept in water to prevent cracking and drying out and about 110 beads will be made. The free enzyme will not take so long to prepare. The enzyme itself, Amylase had a concentration percentage of 0.125%, which is quite strong, to my knowledge. Judging from previous experiments, this has been known to work quickly, so small time intervals will be used in order to get a wide range of results. In order to keep this experiment a fair test, the free enzyme must be diluted to the same extent as the Immobilised enzyme, so 10cm� of enzyme was added to 20cm� of distilled water. Measuring cylinders will be used to measure these amounts. For all catalysis reactions, there must be a substrate for the enzyme to react with, and in this experiment, Starch was chosen. ...read more.


This would then also mean that there would be too much enzyme and all the enzyme molecules would start colliding with each other and not the substrate. Plus, there would be more Maltose being produced and that also would get in the way of the enzyme molecules. If this were to be observed, then you would expect a graph like this one; Point A shown is where all the substrate, in this case Starch, has been used up, and the amount of product made is the amount of Maltose made. Conclusion: Relating back to my prediction, I predicted that the free enzyme would work quicker, and indeed it did. This was because of the theory in my prediction, about the active site of some of the enzyme being unavailable to the substrate in the immobilised enzyme. The active site of some of the enzyme molecules would have been stuck in the Sodium alginate and would have not been used. Proven by the "Lock and Key" theory, there would have been no reaction with the enzyme molecules if the active sites were not available. The free enzyme also worked better because of its ability to float around the sample, leading to the collision rate between the enzyme and substrate being high. The immobilised enzymes would have had to wait for the substrate to reach it, hence the collision rate being low. Evaluation: There are quite a few possible errors in the method previously shown. The experiment should have been repeated more than once. The experiment was planned to be repeated three times in order to get a wide range of results, and to eradicate any anomalous results, an average would have been taken. This would have been done if given more time. The 2cm� of immobilised enzyme is also another area that an error could have occurred. 37 beads were used in order to get a rough 2cm� in a measuring cylinder. ...read more.


Only to a certain amount of heat though, the immobilised enzymes are not invincible, but they are more tolerant to heat than the free enzymes. This could be useful in batch production of Maltose, or indeed any other substance that requires an enzyme. Immobilised enzymes are used in Fomenters. The hotter it is the faster the enzyme works, until optimum, but if the optimum is slightly hotter, then they can work faster. Evaluation: This experiment was a bit more controlled than the previous one, yet there was still room for mistakes. Again, the 2cm� of immobilised enzymes could have made this an unfair test. The solution to this problem is in the previous Evaluation of the last experiment. That being measuring the 2cm� of enzyme out first, and then making them into beads. In both experiments the p.H. was not checked during the experiment. This should have been checked to ensure that the p.H. had not changed. This is only a precaution and does not need to be done as the Buffer solution was added. The anomalous result in the experiment could be caused by the lack of enzyme in the 2cm� as they may have been 36 beads instead of 37. Its possible that it could have got infected with bacteria, and this would have bred in the test tube, seeing as it was just over optimum. The bacteria could have got in the way of the enzyme and substrate and caused the reaction to take longer then necessary. Again I say that it is a possibility. Human error may also have caused the anomaly. Maybe too much starch was added to the test tube, or too much Buffer solution. The only way to get rid of this is to repeat the experiment 3 times and then take an average. This would have been done if given more time. Overall, the free enzyme works quicker than the immobilised enzyme at optimum conditions and the immobilised enzyme works better than the free at higher temperatures, exceeding optimum. ...read more.

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