The Effect of Substrate Concentration on Enzyme Action

The Effect of Substrate Concentration on Enzyme Action
The Effect of Substrate Concentration on Enzyme Action Aim: To investigate the effect of enzyme concentration on the rate of reaction the enzyme controls, using amylase and starch. Introduction: An Enzyme is a protein, which is capable of starting a chemical reaction, which involves the formation or breakage of chemical bonds. The feature of enzymes are to speed up rate of reaction, it lowers activation energy, denatures if the temperature is too high and is a biological catalyst. Enzymes are very specific they won?t catalyze just any old reaction, only those which are suited for the enzyme. This selectivity is because of the essentially fixed shape of the place where the molecules must get together within the enzyme?s reaction site in order to get close enough to form a bond Bacteria too, must have function enzymes available. Being only a single cell with only a single chromosome, bacteria is quite astonishing in their ability to make structural and functional components of all of the things necessary for the cell to stay alive.
As you see could see in the above diagram of an Enzyme the active site is region of an enzyme where substrates blind. Enzymes and substrates do not react with each other, they hold each other together. Amylase breaks down a chain of glucose dividing them into twos, producing maltose. The Benedict?s test allows us to detect the presence of reducing sugars. Starches are no reducing sugars. The copper sulphate (CuS04) present in the Benedict Solution reacts with electrons aldehyde a group of reducing sugars to form cuprous oxide, a red/brown solution. The final colour of the solution depends on how much precipitation was formed, and therefore gives an indication of how much reducing sugar was present. Hypothesis == I predict that the more concentration of amylase there is added to the solution, means the more number of particles there would be. This would mean the number of collisions would increase. This would mean, as the concentration of amylase increases, the time taken for the starch and amylase to React decreases. Also, I have divided my Experiment into the following five categories: 0.5%, 0.75%, 1.0%, 1.5% and 2.0% of amylase. I have decided to choose these amounts of amylase for a specific reason, for my experiment 1.0% of amylase, the number of collisions should double, as well as for 2.0% of amylase, the number of collisions should be double of 1.0% of amylase. So overall, my prediction is that the experiment 2.0% of amylase should have 4 times the amount of collisions than 0.5% of amylase. Equipment used ====== - Boiling Tubes X 5 ? to conduct experiments in - Test Tube rack ? to hold up the test tubes - Large Syringe ? to measure 5cm³ of starch - Small Syringe ? to measure different amounts of Amylase - Pipette ? to measure precisely the volume used in each experiment is the same - Iodine Solution ? used as a test for starch - Benedict Solution ? used for reducing sugars (glucose, maltose etc) - Spotting Tile ? Used to test for starch - Stop Clock ? to time how long it takes for the starch to become negative - Amylase ? 1cm³ of amylase at different concentrations: 0.5%, 0.75%, 1.0%, 1.5% and 2.0% - Starch ? 5cm³ used in each experiment Method -?- First off all I will gather all equipment, then take a large syringe and measure 5cm³ of starch and place in a boiling tube. After that I?ll take a small syringe and measure 1cm³ of amylase depending on the following concentration: 0.5%, 0.75%, 1.0%, 1.5% or 2.0%. Next, place iodine is a spotting tile. Two drops per spot. Add amylase to starch and begin the timer. Add an equal mixture to the spotting tile every 30 seconds. When the solution turns from black to light brown, record the time it took for the starch to become negative. Fair Test For each experiment, the stop clock needs to begin as soon as the starch mixes with the amylase, to make it a fair and reliable test. Otherwise if the stop clock is started halfway through the experiment, the results become unreliable and variable. Also, the concentration in each experiment should be measured precisely, measuring the exact concentration of the amylase by using a small syringe and a pipette. If not, the results will be inaccurate and there will not be a clear pattern showing how the concentration of amylase affects the rate of reaction the enzyme controls. Also, another point to bear in mind is to not measure out the two solutions in the same measuring cylinder before the experiment, otherwise the solution will have already mixed thereby making it very unreliable. Another way of making sure that the experiment is fair is to ensure that the temperature surrounding the investigation is the same for each experiment. Also if the amount of amylase was more, it will affect the experiment, because the more amylase in the experiment, the more particles, meaning it will affect the Rate of Reaction the enzyme controls. Safety Test -???? - When dealing with acids in a lab, you should always wear goggles in case there is a spillage, which could be very harmful and cause the Acid to burn your skin and eyes. - Another basic rule when in the lab working with Acids is, never run around. This floor could be very slippery and if your holding acids you could harm yourself and others working nearby. - Always tie your hair when doing practical experiments, especially when working near a Bunsen burner. You could get distracted for a moment and the situation could be fatal. - Amylase is a digestive enzyme. It can be dangerous if it gets on wounds. Therefore, avoid direct contact with it. - Make sure that when you put iodine it does not get on your clothing as it can leave permanent stains. - Do not swallow or drink any substances, as it can be very harmful. - Do not eat or drink during any part of the experiment. - When using matches, be sure to handle them with care. - If a bottle of a chemical is broken on the floor immediately locate and contact the lab technician. - After the experiment ensure to wash your hands thoroughly before touching any part of your body. - Do not run around in the laboratory and make sure all bags are underneath the tables. Observation -???? Here are the Results I have collected from my experiment in the form of a table: Amylase % Time Taken (sec)c 0.5 84 0.75 24 1.0 17 1.5 18 2.0 10 It is clear to see that there is a steady decrease in the amount of time taken for the solution and the reaction speeds up. Conclusion From the Investigation I have conducted, it is clear to see from my results that: there is a steady decrease in the Time taken for the Amylase to react with the starch, when the Amylase concentration is increased. This is mainly because when the concentration is increases, there is more molecules to react with, meaning they are more likely to collide with each other. This would mean as the concentration increases the amount of Time Taken for the molecules to react Decreases, speeding up the reaction. In my Hypothesis, I clearly stated that ?as the concentration of amylase increases, the time taken for the starch and amylase to react decreases.? This proves that my prediction is correct. Another factor that can also affect my results though is the temperature. If the temperature changes while doing the experiment then I have not kept the variables constant meaning my experiment is not accurate. Evaluation As shown in my Graph, I do have a few anomalies, which prove that I may have had a few problems when performing my experiment: When measuring the amount of Acid in the measuring Cylinder, I always brought the Cylinder to my eye level, thus making the test unreliable and inaccurate. To make my experiment more accurate, I should put the measuring cylinder on a flat surface and see if it the correct amount. Another problem I encountered was with the stop clock, which made the test very unfair as each time I started the experiment I kept pressing the wrong button, and by the time I got it right, the experiment had already started. The test could have also been more accurate if I repeated the experiment a couple more times, thus getting a more accurate result or even by making the temperature go up by 2º, where I would have been able to see where exactly the Rate of Reaction begins to speed up and at what Temperature. Also, a burette could have been used to measure the volume more accurately, to give a more precise volume. This test may have also been unfair due to the lack of accurate equipment. Overall, I am pleased with my results and findings, and have a better knowledge of carrying out experiment in the further.

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