An investigation into the effect of pH on the starch hydrolysis of fungal and bacterial amylase

Authors Avatar

An investigation into the effect of pH on the starch hydrolysis of fungal and bacterial amylase.

Hypothesis

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.

Join now!

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 ...

This is a preview of the whole essay