Hypothesis
My hypothesis is that:
‘The greater the pH the higher the activity of bacterial amylase in starch hydrolysis than fungal amylase‘.
Fungi and bacteria are both forms of amylases and are used in feeding processes.
As mentioned above bacteria are prokaryotes they are very small organisms with no true nucleus and a unicellular form and occur by clumping together in characteristic patterns forming chains but the cells can also be individual cells and have to be seen under microscopes as they are very small. They can be found in most environments such as dust, air, soil, water and living materials. Together with fungi they play an important role in vital activities with organisms in decaying of recycling of nutrients from organic matter. Some bacteria can inhibit in many environmental extremities of pH 1-11 in order to maintain constant cytoplasmic pH ranges.
Fungi are eukaryotes which are much bigger cells with membrane bound nuclei and organelles. They compose of mildews, rusts, smuts, puffballs, mushrooms, yeasts, stinkhorns, toadstools etc. They grow on dead matter, parasites or dissolved nutrients through their cell walls. It has a fungal body made up mycelium with parts of hyphae( fine, thin, long threads) which form even longer branching chains of fungi cells. Fungi usually grow best at slightly acidic pH of around 5-7 and so can reproduce at this range.
I have chosen bacterial amylase that work best at higher pH values because they can be found in a range of different habitats and if this is so it can tolerate the extremities in pH and so would have a larger optimum pH range.
Generally any enzyme works best and faster at a certain optimum pH range, under constant temperature. Some work in more acidic conditions such as pepsin in the stomach will need to work best at pH 2 as it is working with hydrochloric acid, and others in alkaline conditions.
Increase in temperature creates a bigger kinetic movement across the molecules and so having a greater chance of them colliding with each other. However enzymes also work at optimum temperatures before they become inactivated (denatured). If one is working with pH the temperature has to be kept constant as it is a factor that can also affect enzymes.
The optimum pH is the maximum rate of reaction that occurs in the range. When pH is altered below or above the optimum the activity is decreased or becomes denatured. As pH is reduced it becomes acidic and an increase in H+ ions which increase the amount of positive charges. Alterations in pH change the ionic charges of the acidic and basic groups and therefore mutate the R-groups in the amino acids which disrupt the ionic bonding which help stabilise the specific shape of the enzyme. The pH change therefore leads to the alteration of the enzyme shape and also the active sites in which substrates are broken down, causing the enzyme to denature.
A graph showing enzyme activity against pH
I am going to investigate the effect of pH of the enzyme activity of fungal and bacterial amylases using various pH and using it to hydrolyse starch and testing it using potassium iodine solution for starch present under timed conditions with these two different amylases separately.
The presence of starch can be discovered using iodine dissolved in potassium iodide which is a orangey brown colour and changes to a dark blue-black colour when starch is present. The experiment is to investigate the effect of pH on the two types of amylases on its activity. The relative activity of the amylases is noted by the time taken for the starch substrate to be broken down resulting in a colour that is no longer a blue- black colour when tested with the iodine solution. This is known as the achromatic point.
Key variables
In order for the experiments to be fair and reliable each time the following must be taken in consideration:
starch concentration and volume will be the same in all experiments.
Amylase concentration and volume will be the same in all experiments.
The same concentration and drop size of iodine will be used on the spotting time to determine starch presence.
Buffer solutions would be used to avoid any changes in pH differences
The iodine solution would be used as a guide to determine the end point colour.
Other volumes of liquid would measured accurately using measuring cylinder and pipettes.
All this would be timed using a stop watch
Apparatus
0.1% fungal amylase and bacterial amylase
Potassium iodine solution
1.0% starch solution
White spotting tile
10 clean test tubes
Test tube rack
Buffer solutions ranging from pH 4-8
Pipette
Measuring cylinder
And stopwatch.
Method
Take a test tube rack and place five clean test tubes each for the fungal amylase and bacterial amylase and place a sticker with a different pH of 4-8 so you do not get confused.
Prepare the buffer solutions by diluting the acid or alkali of certain concentration (mol dm ) with 100ml of water. Use a universal indicator to check the pH.
Pipette 1cm of either fungal or bacterial amylase decide which one you are going to do first in each of the five test tubes. Then take a pH solution of 1 cm and place in one of the test tubes with its correct label. Time this for ten minutes. Whilst waiting place equal drops of potassium iodine solution in different places on the spotting tile. After ten minutes, add 5 cm 1.0% starch solution.
Now for every thirty seconds for five minutes, pipette a bit of the solution and place it on a clean spot of iodine solution and record the results. + is dark blue-black, +/- if dark brown, and - is no colour change.
Repeat this with the other pH solutions and the other amylase enzyme.
Risk assessment/ Safety precautions
Amylase:
It is brought in powdered form and so can causes respiratory problems if inhaled in a poorly ventilated room. It may cause risk of eye damage if contact with eye. On skin contact can causes cracked skin or eczema.
To avoid this wearing eye protection and gloves to be safe.
Buffer solutions:
Handle with care wash with water if comes in contact with skin. Protect from eyes as it can be irritant. Avoid by wearing gloves and eye protection.
Iodine in potassium iodine solution:
It can be harmful if inhaled, swallowed or absorbed by skin. If absorbed by skin or comes in contact with eyes it can be irritant. It causes gastrointestinal discomfort if swallowed or respiratory problems if inhaled.
Washing spotting tiles thoroughly under running water and clean any spillage.
WEAR SUITABLE PROTECTION WHEN HANDLING CHEMICALS.
Starch:
Starch has very little harm or risk
USE ALL SUBSTANCES FROM CLEARLY LABBELED CONTAINERS WITH LIDS .
Results
Fungal amylase:
Test 1
Test 2
Key: + blue/ black, +/- dark brown, - iodine colour (orangey brown).
Bacterial amylase
Test 1
Test 2
Key: + blue/ black, +/- dark brown, - iodine colour (orangey brown).
For me to compare the effect of pH on the amylases, I calculated the overall rate of reaction by: 1/ time (sec).
Fungal amylase and bacterial amylase
Conclusion
Trends and patterns
From the graphs it is seen that the bacterial amylase only began to hydrolyse starch at a higher pH of 8 whereas fungal amylase worked at a range of pH of 5-8.
From the raw data it shows that bacterial amylase is significantly less active at pH of below 8 compared to the fungal amylase and begins to activate or works at pH of 7 or more in the alkaline range.
Fungal amylase began to hydrolyse starch at pH 5 with a slight change in colour of dark brown however, it is not until 120 seconds where the starch is completely broken down with no change in iodine colour representing that starch is not present. This is seen till the end of 300 seconds.
As compared to bacterial amylase activity shows that in the from both the graph and table, the activity of break down of starch is entirely inactive in overall rate of reaction of 0 at times of 30-300 seconds of pH 4-7. These is a considerable change in reaction at pH 8 where there is some hydrolysis of starch as seen by the colour noticed of dark brown.
However, due to only using a limited number of pH buffer solutions in the experiment it was impossible to work out the optimum pH ranges as there was a limit in basic pH solutions.
Explanation of results
Enzymes are globular proteins that work at specific conditions. They are made up of a tertiary structure which consists of polypeptide chains and many folding which make its shape. The folding of peptide chains are held together by ionic, hydrogen, and disulphide bonds. The frame of the end enzyme in this case amylase enzyme of either fungal or bacterial form is important to activity of enzyme as it affects the way in which the substrate will fit into the active site and therefore enable the substrate to break down which lead to the formation of products. The greater the size of the 3-D structure of the protein, the peptide chains that are held by the bonds will be affected due to alteration in pH creating severe changes in the R-group of the enzyme which codes its particular function of the job it has to carry out. When there are changes in the R-group this
disrupts the ionic groups if pH was high, as mentioned before, will cause modifications in the active site preventing the substrate fitting comfortably and break down or would damage the enzyme completely can causing it to denature or in cases of lower pH, it will be unsuitable for the enzymes to work and therefore will remain inactive.
As seen from the results the bacterial amylase was inactive as pH of 4-7 which proves my hypothesis that ‘ the greater the pH the greater the activity of bacterial amylase on starch hydrolysis compared to fungal amylase‘. A reason for this may be in the nature of the amylase; bacterial amylase which are prokaryotes work better in basic (pH above 7) conditions. Bacteria are prokaryotes the early forms of cells. They are unicellular as they do not a contain membrane bound nuclei or organelles and depend entirely upon living things. Bacteria digest organic matter releasing organic acids in to the soil. This can initially lower the rate of pH. In anaerobic conditions where enough oxygen is present for the break down of organic matter, these acids are neutralised as decaying carries on allowing the pH to change to between 7 and 8.
In contrast fungal amylase belong to the fungi kingdom which are like plants but lack chlorophyll and work best at pH 5-7 and so need these conditions in soil to reproduce and react more in these acidic conditions in which they gain their nutrients from and use as their food production.
Evaluation
There were a few limitations in the experiment which could be taken into consideration if I were able to do again. Firstly, the important one is making judgement of accurate final end point. Determining the colour of iodine in slower reactions of blue-black and dark brown was difficult in some cases. In the end of reactions on bacterial amylase was difficult to determine the iodine colour whether or not they may be an change. This method may be inaccurate as slight colour change is taken on own judgement.
Secondly, another limitation is that even though the enzymes of the same concentration was constant in the experiment, the powdered enzymes was not reliable as it was a raw preparation as the enzymes might of contained different active ingredients with different active rates within the concentration affecting the overall activity of both enzymes making the comparison harder to see with each other.
The overall experiment could of been more and reliable accurate if further tests were done, like mentioned in the conclusion, there was a limiting amount of pH buffer solutions used. There was not equal amounts of basic pH compared to acidic pH.
I would of done a preliminary study to get a more understanding of the experiment such as looking at different concentrations of starch, different concentrations of amylase enzyme etc which would help in explaining in greater depth the effect of pH.
In future if I had more time more the experiment, I would of liked to of worked with even more ranges of pH e.g. pH 7.2, 6.5 etc . I would also of like to work with a bigger range of fungal and bacterial amylases in subject to availability.
