The effect of temperature on the hydrolysis of starch using amylase extracted from barley.

Sasha Caddy, RM11. 20/01/04

The effect of temperature on the hydrolysis of starch using amylase extracted from barley

Interpretation of results:

Enzymes are a class of proteins that catalyse chemical reactions, which increases the rate of a metabolic reaction. Most enzymes are specific, working on a particular or class of reactions. In this case I am using an enzyme known as amylase (a group of enzymes which convert starch to sugar), which is an important metabolic enzyme. Amylase is found in various parts of the body including the saliva of the parotid gland and the pancreas, e.g. ptyalin, which aids in the digestion of carbohydrates by speeding up specific digestive processes taking place from the mouth to the small intestines. However, in this experiment we are using amylase which has been extracted from barley. The function of amylase is to catalyze (to modify the rate of a chemical reaction by catalysis) the hydrolysis (decomposition of a chemical compound by reaction with water) of starch into glucose. Starch is a mixture of two compounds; amylose and amylopectin, both of these molecules are polymers which contain a large, variable number of a-glucose molecules linked to each other by condensation. Amylase acts on starch, which is a polysaccharide (a class of carbohydrates; starch, consisting of a number of twenty-five monosaccharides) and breaks it down into maltose, a disaccharide. A disaccharide is defined as any class of carbohydrates; maltose, that yield two monosaccharides upon hydrolysis. The disaccharide sugars; maltose, lactose, and sucrose, have the empirical formula C12H22O11. When treated with enzymes, the disaccharides combine with one molecule of water and split into two molecules of monosaccharide hexose sugars, e.g. maltose splits into two molecules of glucose when treated.

In order for amylase to continue working at its best, the body needs to keep within several degrees of 37 C (an optimum temperature for most enzymes), as enzymes must work in mild conditions of a cell in the body. Chemicals which are changed by enzyme-catalysed reactions are known as the substrates of that enzyme, which fit into the active site (where the reaction takes place) of the enzyme, in a lock-and-key mechanism. The products of the reaction then leave the active site, which frees it up for more similar reactions to take place. If our body heat exceeds further past 37 C our cells become impaired or permanently damaged, this damage is irreversible to the molecular structure of the enzymes due to the velocity with which the atoms move about. This is because the structure of the an enzyme vibrates so much that some of the bonds holding the tertiary structure together break (especially hydrogen bonds as they are weak). So now the enzyme starts to lose its globular shape, because of this the substrate will no longer be able to fit into its active site. In other words when the enzymes become denatured, there is a major change from the native state to another state without the changing of the primary structure, this usually leaves the enzyme without its catalytic functions. At a temperature of approximately 100 C amylase becomes denatured. Whereas, if our body heat was to descend below 37 C the metabolism decreases without permanent damage until ice crystals form in the cells. Meaning the enzymes are inactivated, not denatured (even at extreme low temperatures, such as 0 C) and once the temperatures increase, they will regain their function.

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From the first graph which shows the percentage transmission from the colorimeter (a device which provides an indication of how deep a colour is, and could measure the index of concentration of the samples) at minute intervals at different temperatures; 15 C, 25 C and 35 C, there is a trend and pattern. This trend and pattern is that the lower the percentage of transmission from the colorimeter, the less light getting through, this means that there is a high concentration of starch (mg). Although, as time increases more and more of the substrate (starch) is being broken down into maltose so there is an increase of transmission from the colorimeter, meaning more light is passed through the solution. For example, at 35 C and at 0 minutes there is 1% transmission from the colorimeter, meaning that only 1% of light can pass through the solution because there is 465mg of starch (shown by the 'Starch Calibration Curve'). As time increases to 20 minutes there is a 40% transmission from the colorimeter meaning there is 70mg of starch concentration left in the solution because it has been broken down by amylase at a high activity rate. The biological knowledge to support this trend and pattern is the kinetic theory; when a substance is heated, its molecules is being supplied with kinetic energy, so they move around faster. In this experiment, as the temperature rises from 15 C to 25 C to 35 C, there is an increase in the number of collisions between the active site of the enzyme and starch molecules and with more energy. This causes them to react more efficiently as this results in more enzyme-substrate complexes and in turn the formation of more products. At low temperatures e.g. 15 C, the molecules will not collide very frequently and the starch will not be broken down as quickly. This shown on the graphs at 15 C and at 0 minutes there is 0% transmission from the colorimeter, meaning that 0% of light can pass through the solution because there is 500mg of starch (shown by the 'Starch Calibration Curve'). As time increases to 22 minutes there is a 15% transmission from the colorimeter meaning there is 160mg of starch concentration left in the solution. This is because it has been broken down by amylase at a slow activity rate, so there is a higher concentration of starch left compared to the 25 C (120mg) and 35 C (70mg) results.

From the second graph; 'A graph to show the milligrams of starch at minute intervals at different temperatures', it shows that with time, the starch concentration is decreasing for each temperature that is being tested. This graph shows an exponential decay curve of the amount of starch concentration broken down for every x minutes, therefore the substrate will not totally be broken down. This reaction is not a equilibrium reaction because as the starch concentration decreases the enzyme finds it increasingly difficult to find enough substrate to act on.

From my results, I can conclude that between temperatures 15 C - 35 C, the efficiency of the enzyme increases with temperature. Therefore, the graph shows that 35 C is the optimum temperature because at the end of the experiment (at 20 minutes), the solution has a high percentage of transmission (40%) meaning 70mg starch left. So the amylase is breaking down the starch most effectively at 35 C due to the more light passing through from the colorimeter. These figures show that at 35 C the hydrolysis of starch using amylase is a lot more active, because the body temperature is around 35 C and enzymes such as amylase, are designed to work at this optimal temperature. So at 35 C maltose is formed a lot faster than at 25 C and 15 C. Whereas, at 15 C and 25 C the graphs show that the activity of the amylase is working at a much slower rate, therefore unable to break down as much of the starch in approximately 20 minutes. This is shown by a less percentage from the colorimeter, which does gradually increase over more time when more milligrams of starch is broken down into maltose.

Evaluation of practical work:

The experiment worked well overall, proving that the optimum temperature of the amylase used in this experiment was around 35 C. The results are sufficiently accurate as each set of results align almost a perfect curve, and they were taken at timed intervals far enough apart so that the readings are clear from each other.

In this practical procedure the results could have been influenced by main sources of errors such as:

The apparatus could have been improved as the water baths used were not all at the exact temperatures required, and each water bath possibly contained different amounts of water. If better quality water baths had been used and more time was issued to ensure that each of the three water baths had exactly the same amount of water and was at the exact temperature required, more accurate and reliable results would have been achieved. This also could have been achieved by repeating the experiment for each temperature more than twice and then calculating averages of the two sets of results.
Also the use of a colorimeter could have altered the results of the colorimeter readings when it was set at 100% with a test tube of diluted iodine by a member of the group. To improve this we need to have used a photospectrometer which is a device that can stop the fluctuation of these percentage transmissions resulting in precise and accurate results.
We should have performed the experiment at intervals smaller than 10 C, so that we used a wider range of temperatures e.g. 10 C - 70 C. At this temperature range I would have been able to see whether at the lowest temperature if the enzyme; amylase, could function at all effectively and that amylase would possibly denature at 70 C and definitely at a temperature above 70 C. Proving that above 70 C the amylase is denatured therefore no longer catalyzes the hydrolysis of starch, which is broken down into maltose. Conducting the experiment at 10 C intervals between these temperatures would have obtained a sufficient number of distinct results.
I think that the pipettes used were another main error. More accurate results could have been obtained by cleaning the pipette between each reading, or using a new pipette each time, although this couldn't practically happen. There was always some solution left over in the pipette from the previous solution, whether this was iodine solution, distilled water or when we were extracting the reaction medium and placing it in the diluted iodine solutions. Another problem with the pipettes is that when the reaction medium was extracted and clearfully put into a diluted iodine solution, during this time the amylase was acting on the starch while this solution was in the pipette. This made the timings recorded slightly out, although this effect may have been lessened with the temperature at 35 C as the mixture was cooling down to room temperature in the pipette.

Also we could have possibly swirled the enzyme extract and starch solution together in the water bath so that the substrate and enzyme could mix and the molecules collide. A solution to this whole experiment would have been to automate (convert to a automatic operation) the whole system. This would have allowed a sample of the mixture to be automatically taken every minute or possibly more frequently, and the concentration of the starch stored onto a computer. Carrying out the experiment like this would have solved any inaccuracies in timing, which may not have always been exact when using a stop clock and someone watching the time. This way it would have also removed any human errors e.g. the test tube not being wiped properly before being placed into the colorimeter or didn't shake the reaction medium and diluted iodine solution together enough/too much etc. So if the experiment had use of better apparatus and stricter conditions, my results would have been plotted onto a graph and a more clear and accurate curve would have resulted.

In this experiment there were no anomalous results which can be shown from the graphs.

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Adam Roberts

17th of Sep 2013

**** This account includes detailed background theory and good discussion and evaluation sections. However, in places biological terminology might have been used more carefully.

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