An Investigation into the Effect of pH on the Starch Hydrolysis of Fungal and Bacterial Amylase

An Investigation into the Effect of pH on the Starch Hydrolysis of Fungal and Bacterial Amylase
The optimum pH of fungal amylase is lower than the optimum pH of bacterial amylase. Variables In order to keep results reliable it will be important to ensure that there is only variable. This will be pH. This means that all other things must be kept constant. In order to achieve this certain precautions need to be taken. Temperature will be maintained by putting the agar plates in an incubator set at a constant of 20ºC; this will be checked with the use of a thermometer. This is important because an increase in temperature will increase kinetic energy therefore speeding up the rate of reaction. High temperatures could even denature the amylase. Low temperatures will decrease the rate of amylase activity because the substrate and enzyme molecules have less kinetic energy and move more slowly, collide less often and react more slowly.
The same concentration of bacterial and fungal amylase will be used (0.1%). It is known that a higher concentration will cause a faster reaction rate, this is because there will be more active sites for the substrate to react with. To keep the concentration at a constant the enzyme will be weighed out on a top pan balance to an accurate degree, and dissolved in 100cm³ of distilled water. The rate at which the enzyme functions depends partly on the concentration of enzyme molecules. The greater the concentration of amylase, the more active sites there are and so the more chances there are of collisions between the substrate and active site. The same volume of buffer and amylase needs to be used in each hole on the agar plate. A 1cm³ syringe will be used, as this can dispense within a 0.005cm³ accuracy. Also in order to keep the diameter of the hole constant a cork borer of diameter 5mm will be used. It is difficult to control the depth of the agar so the depth will be measured and used in the calculation of the rate. All the agar plates need to be left for the same length of time to react with the amylase before they are flooded. To keep this constant they will all be flooded at the same time. The length of time suitable to give the most accurate results will be determined by the pilot experiment. Plan The activity of both fungal and bacterial amylase will be measured by determining the rate at which starch is hydrolysed. With the use of a cork borer, six holes will be cut into ten starch agar plates. Half of the plates will have holes filled with 0.05cm³ of buffer solution and 0.05cm³ of fungal amylase (using a 1cm³ syringes). The other five plates will have holes filled with 0.05cm³ of bacterial amylase and 0.05cm³ of buffer. Five buffers will be used, pH 2, 4, 6, 8 and 10. There will be separate plates for each pH, for both the fungal and bacterial amylase. The agar plates will be left in an incubator set at 20ºC to keep temperature constant and will be left for a period of time which will be determined through a pilot experiment. Then the agar plates will be flooded with iodine. The iodine will then stop the amylase from digesting anymore starch. A blue/black colour indicates where starch is still present and areas where the agar is colourless will show where the amylase has digested the starch. These areas are expected to be circular around the holes in the agar plates. The diameters across each of the clear areas will be measured in mm using a compass. The depth of the agar will also be measured. Pilot experiment Before carrying out the investigation a pilot experiment needs to be carried out in order to determine the most appropriate length of time in which to leave the agar plates, and to test the planned method of data collection. This is necessary because if they are not left for a long enough time then the differences between the volumes of the starch area digested will be minimal and results could be less reliable. However if they are left for too long then the colourless areas from each of the six holes could merge into one another, this could also make results inaccurate. In the pilot experiment eight agar plates will be used with two different buffers, pH 4 and pH 8. Half of the plates are to have holes containing bacterial amylase and the other half fungal amylase. Some of the agar plates will be left for seven hours and then flooded with iodine, and the others will be left for twenty-four hours, then flooded. The diameters across the colourless areas are to be measured using a compass. One of the agar plates will also be used to test the volume of liquid which each hole is able to hold, without it overflowing. I have decided to use amylase with a low concentration, so that the reaction will not occur too quickly, because if they are left for some time, this too could cause the colourless areas to become overlapped, and it will be more difficult to determine a suitable length of time in which to leave the agar plates. Apparatus for pilot experiment
pH 4 buffer
pH 8 buffer
0.1 g 100cm3 bacterial amylase
0.1 g 100cm3 fungal amylase
stopwatch
3 syringes (1cm³)
iodine
compass
8 starch agar plates (1 g 100cm3)
cork borer
ruler
Risk assessment In order to maintain a safe environment and ensure that results are not wrongly affected, some general safety procedures need to be carried out. This is a low risk procedure, with no glassware involved. However making the agar plates involves dissolving agar in boiling water. For safety reasons these plates will be made by the lab technician. In touching the agar plates bacteria could be transferred from hands, therefore gloves will be worn throughout the experiment. Some of these bacteria may be pathogenic (disease causing) and they could be cultured on the agar plate. Also the buffers need to be used with care, because strong acids or alkalis could be harmful. Iodine could also be dangerous as it is an irritant, for this reason safety goggles will be worn to minimize the risk or contact with my eyes. After the experiment it is important that the agar plates are autoclaved, in order to kill the bacteria and fungi. This involves heating the plates to a high temperature of about 120ºC and for safety reasons (due to the high temperatures involved) this process will be carried out by the lab technician. Pilot results- Table 1 The effect of time on the starch hydrolysis by fungal amylase in agar Time left before flooding with iodine (hours) pH Diameter of hydrolysed starch (mm) Depth of agar (mm) 1st hole 2nd hole 3rd hole 4th hole 5th hole 6th hole Mean (3.s.f) 7 4 13 14 13 13 14 16 13.8 5 7 8 10 12 10 11 11 10 10.7 4 24 4 26 22 21 20.5 21 24 22.4 6 24 8 23 19 19.5 20 19 20 20.1 7 Pilot results- Table 2 The effect of time on the starch hydrolysis by bacterial amylase in agar Time left before flooding with iodine (hours) pH Diameter of hydrolysed starch (mm) Depth of agar (mm) 1st hole 2nd hole 3rd hole 4th hole 5th hole 6th hole Mean (3.s.f) 7 4 9 9 8 8 9 7 8.33 5 7 8 10 9 9 10 10 10 9.67 5 24 4 19 19 18 17 18 19 18.3 4 24 8 19 19 19 20 19 18 19 5 From carrying out the pilot experiment, I have observed some flaws in my original procedure, and methods of measurement. For this reason I have decided to make some modifications to the way in which the experiment is carried out, and to how the results are recorded. -In the pilot, one of the agar plates was used to work out the volume of liquid that can be fitted into the hole made my the cork borer. This was found to be 0.1cm³. This is a relatively small volume, so in order for there to be a significant volume of both amylase and buffer I decided that 0.05cm³ of each should be used in every hole. Although this is a small volume a 1cm³ syringe can measure this quite accurately. -Looking at the results, it appears that six results for each pH is a justifiable number of repeats. As the mean values give a good representation of the raw results. So I will be doing six repeats for each pH tested (these will be 2, 4, 6, 8 and 10) for both fungal and bacterial amylase. -The depth of the agar was checked to see if it was constant, however, because the plates did not all contain the same depth of agar it was obvious that the results were not reliable. The diameter would usually be a reliable measurement, but if the depths are different, the diameter does not effectively show the relative effect of pH on hydrolysis of starch by amylase. In order for the results to be reliable, I have decided it is necessary to calculate the volume of the area where starch has been digested. The volume of the hole in the agar will be calculated, and this will be subtracted from the colourless area, assuming that both the areas are perfect cylinders and using the equation: Volume of a cylinder=Πr²h Where r is the radius (this is half of the diameter, which I will have measured) and h is height (this will be the depth of the agar). This will be done using the mean diameter of starch hydrolysed for a certain pH and the depth of the agar in that plate. -Lids will be kept on the agar plates, to prevent cross contamination and evaporation whilst they are in the insulating oven. -Referring back to the results from the pilot experiment, it is obvious that there is a much greater diameter in which starch has been hydrolysed after 24 hours. The means are almost double the diameter of those left for only 7 hours of the corresponding pH and type of amylase. None of colourless areas of hydrolysed starch overlapped each other after this length of time, so I have decided to leave the plates for 24hours, to maximize the volumes of starch hydrolysed, in order for there to be more reliable and notable differences between the results at different pH values. -I felt the use of a compass as a measuring tool was slightly unreliable, and did not feel that it was giving the most accurate measurements. Therefore I am now planning to use dividers as I think these will be easier to use and will give more accurate measurements. Revised apparatus
10 starch agar plates (1 g 100cm3)
dividers
0.1 g 100cm3 bacterial amylase
0.1 g 100cm3 fungal amylase
stopwatch
pH 2, 4, 6, 8 and 10 buffers
cork borer
iodine
3 syringes (1cm³)
ruler
Introduction Amylase is an enzyme and is therefore classed as a biological catalyst. A catalyst is a chemical agent that speeds up the rate of metabolic reactions, by lowering their activation energy (which is the energy required to start a reaction), without being used up in the process. It does this by changing the pathway which the reaction takes. Enzymes are site specific, meaning they can only act on a particular substrate. All enzymes are proteins. They have a three dimensional (tertiary) structure that are specific to their function. The structure is held together by hydrogen bonds, ionic bonds and disulphide bridges. During the reaction some bonds are broken and some are made, this allows the reactants to be changed into products. On the surface of the enzyme is a depression called an active site. This is formed from the residues of a small number of amino acids and has a very specific shape that is complimentary to the shape of the substrate. Any factor that changes the tertiary structure of the whole protein can alter the shape of the active site and alter the activity of the enzyme. There are some things that can affect enzyme activity, and in some cases stop activity completely. These include temperature, pH, concentration of substrate and the presence of inhibitors. In this investigation the effects of pH will be tested.pH is a term used to define the concentration of hydrogen ions. Each enzyme has an optimum pH, though many work best at neutral or slightly alkaline conditions. An inappropriate pH can change the active site drastically so that the substrate can no longer bind. This means the reaction will not take place. Therefore a change in pH (whether higher or lower) will effect the rate at which the enzyme will work, because it can have a direct effect on the ionic bonding responsible for the secondary and tertiary structure of enzymes. Resulting in a change in shape of the active site, denaturing it so that its specific substrate can no longer fit into the active site. A rise in pH level means that there is a higher concentration of hydroxyl ions (OH-) which are alkaline, and the active site repel the substrate. If there is a low pH there will be an increase in hydrogen ions (H+) these are acidic and also repel the substrate. This therefore causes a drop in reaction rate. It is very important to control the other variables that can affect the rate of amylase activity so that the only factor that affects the activity of the amylase in this investigation is the pH. The enzymes being used in this experiment are fungal and bacterial amylase. Both enzymes hydrolyse starch molecules into alpha glucose molecules by breaking the alternate 1, 4 glycosidic bonds, producing maltose. Bacteria are prokaryotic, unicellular organisms. They can be found in water, air and soil, it is because bacteria can survive in such a wide variety of habitats that pH is suspected to have less effect on bacterial amylase activity. Fungi are eukaryotic multicellular saprobiontic microbes, which vary in size but are larger that bacterial cells. Fungi feed on dead organic matter or parasites. Fungi are better adjusted at living in acidic environment, including citrus fruits. Therefore it can be suspected that fungi have a low optimal pH. Hypothesis The optimum pH of fungal amylase is lower than the optimum pH of bacterial amylase. Null Hypothesis === There is no difference between the optimum pH of fungal and bacterial amylase. Method The activity of both fungal and bacterial amylase was measured by determining the rate at which starch was hydrolysed. With the use of a cork borer, six holes were cut into ten 1% starch agar plates. Half of the plates had holes filled with 0.05cm³ of buffer solution and 0.05cm³ of fungal amylase (using a 1cm³ syringes). The other five plates had holes filled with 0.05cm³ of bacterial amylase and 0.05cm³ of buffer. This ensured that there were six repeats for each type of amylase and pH, having done these repeats mean Five buffers were used, pH 2, 4, 6, 8 and 10. There were separate plates for each pH, for both the fungal and bacterial amylase. The agar plates were covered will lids to prevent evaporation and cross contamination. They were left in an incubator set at 20ºC to keep temperature constant and left for 24 hours. Then the agar plates were flooded with iodine. The iodine stopped the amylase from digesting any more starch. Areas with a blue/black colour indicated where starch was still present and areas where the agar was colourless showed where the amylase had digested the starch. These areas were circular around the holes in the agar plates. The diameters across each of the clear areas were measured in millimeters using dividers, and a ruler. The depth of the agar will also be measured, by inserting a pin in the agar until it touched the bottom, then withdrawing the pin and measuring the length of the agar covered area against a ruler. Results A table to show the effect of pH on the starch hydrolysis of fungal amylase, when left for 24 hours- Table 3 pH diameter of hydrolysed starch (mm) depth of agar (mm) 1st hole 2nd hole 3rd hole 4th hole 5th hole 6th hole mean 2 21 22.5 21.5 22 21.5 21 21.58 4 4 21.5 21.5 21.5 21.5 22.5 22 21.75 4 6 22 23 23 23 23 23 22.83 4 8 20.5 21.5 20.5 21.5 21 22 21.17 4 10 21.5 20.5 21 21.5 21 21 21.08 4 A table to show the effect of pH on the starch hydrolysis of bacterial amylase, when left for 24 hours- Table 4 pH diameter of hydrolysed starch (mm) depth of agar (mm) 1st hole 2nd hole 3rd hole 4th hole 5th hole 6th hole mean 2 16 17 17 16.5 16 16 16.42 4 4 19 19 18 18.5 19 19 18.75 3.5 6 23 22 21.5 22 21.5 22 22.00 3 8 19 19 19.5 20 19 19 19.25 3.5 10 18.5 18 19 17.5 18 17 18.00 4 A table to show the effect of pH on the mean volume of starch hyrdrolysed by bacterial and fungal amylase, when left for 24 hours- Table 5 mean volume of starch agar hydrolysed (mm³) to 2 decimal places pH by bacterial amylase by fungal amylase 2 1384.52 768.16 4 1407.65 897.22 6 1559.40 1081.49 8 1329.00 949.94 10 1317.94 939.36 Analysis Results obtained from this experiment show some obvious trends. Graph 1 shows the effect of pH on the mean volume of starch hydrolyzed by both fungal and bacterial amylase. The shape of the graph is typical for enzyme activity with a variable pH. Both types of amylase have increased starch hydrolysis between pH 2 and pH 6, then starch hydrolysis decreases from pH 6 to pH 10. This suggests that the optimum pH for both fungal and bacterial amylase is around pH 6, which would suggest that the hypothesis is not correct. Since both types of amylase have a similar optimum, the Mann-Whitney U test was used to compare the medians of the samples collected for fungal and bacterial amylase. The value for U found using this test was below the critical value at the 5% level. This shows that there is a significant difference between the volume of starch hydrolysed by bacterial and fungal amylase at pH 6 at the 5% level indicating that despite the optimum pH for both fungal and bacterial amylase is around pH 6, there is a considerable enough difference within the data to indicate that whilst bacterial and fungal amylase may have identical active sites, that there are probably differences between the rest of the enzyme. Also from graph 1and the results in table 5 it can be seen that at all pH values tested, fungal amylase hydrolysed a considerably greater volume of starch than bacterial amylase. From table 5 in can be seen that within the pH values tested, fungal amylase hydrolysed the smallest volume of starch at pH 10, a volume 15.5% less than at pH 6. Whereas at pH 2 only 11.2% less was hydrolysed. Indicating that fungal amylase is affected to a greater degree by alkaline conditions. This coincides with the fact that fungi is well adapted to living in acidic environments, therefore the structure of fungal amylase is less damaged by low pH, but in conditions of high pH there is a greater effect on the ionic bonding responsible for the secondary and tertiary structure of enzyme, resulting in a change of shape at the active site. Fungal amylase however, hydrolysed the smallest volume of starch at pH 2. There is a vast difference in the volume of starch hydrolysed between pH 2 and pH 6 of 29%, but only a 13.1% difference from pH 6 to pH 10. This indicates that bacterial amylase is adapted better for alkaline environments, and therefore the structure of the enzyme is effected more by acidic conditions. The fastest rate of amylase activity measured in this experiment was at pH 6 for both bacterial and fungal amylase but it is not possible to know whether this was the actual optimum pH for either enzyme. On the basis of this experiment the hypothesis must be rejected. Evaluation Six repeats were made for each pH value tested, for both fungal and bacterial amylase. Some of the sets of repeats show little variability and therefore can be considered reliable; for example the diameter of starch hydrolysed by fungal amylase at pH 6 has a mean diameter of 21.08mm and all repeats are within 2.8%. All other pH values tested for fungal amylase have repeats within 4%. With bacterial amylase at pH 2 the mean diameter of starch hydrolysed was 16.42mm and all repeats are within 3.4%. However in other bacterial amylase agar plates of different pH, there is a slightly greater variability, indicating that the results may not be completely reliable. For example for with bacterial amylase at pH 10 there was mean diameter of 18.00, with repeats within 5.26%. None of the repeats, for any of the pH values were beyond 5.26% of the mean, but to increase reliability, more repetitions would need to be made for each pH value for both fungal and bacterial amylase. The variability of the results can also seen on graph 1 where the error bars provide the largest and smallest volumes of starch hydrolysed within each agar plate. The greater the size of the error bar, the less reliable the results are. In the graph none of the bars overlap one another this means that there definitely is some variability in my results, however none of the bars, or the data show any significant outliers. In table 3 and table 4 possible anomalies are highlighted in red, these are the results furthest away from the mean. However it is not believed that these results would have any significant effect on the overall results, and they have been included in all calculations. The reason that these anomalies may have occurred is due to lack of control of variables, as this causes the variation in raw results which leads to anomalies. Despite trying to control all possible variables there are a few areas within the plan which could have lead to inaccuracy in the results.

An Investigation into the Effect of pH on the Starch Hydrolysis of Fungal and Bacterial Amylase 6.9 of 10 on the basis of 2811 Review.