A Comparison into how temperature effects the rate of reaction of Bacterial amylase and Fungal amylase.

A Comparison into how temperature effects the rate of reaction of Bacterial

amylase and Fungal amylase.

‘Amylases hydrolyse glycosidic bonds in polysaccharides such as starch…to maltose.’p39 molecules and cells-Nelson advanced science.

They are found in two forms - α and β amylase. Both work by hydrolysis – adding one molecule of water across the glycosidic link.

Hypothesis

My hypothesis is that bacterial amylase will be more resistant to denaturation at temperature exposure (above 40 c) than fungal amylase.

Justification

Enzymes, being proteins, are sensitive to environmental change. Temperature (and pH) can affect the activity of a molecule, as they can change the shape of the enzyme molecule.

As temperature of molecules in solution increases, the more kinetic energy both enzyme and substrate molecules contain, therefore colliding more energetically and frequently, thus increasing rate of reaction. However, after the initial rise in the rate of the reaction, as temperature continues to increase, due to enzymes being made up of protein, they are adversely affected by high temperatures, and often above 45 c many enzymes are denatured.

However, temperature increase, also affects how stable the enzyme molecule is, as precise shape of the active site is essential for catalytic activity. Therefore, a slight change in the shape of the active site, would result in it not being able to combine with its substrate, thus affecting rate of reaction, concluding in the enzyme being de-natured – when it loses its catalytic properties, and is unable to combine with its substrate, due to irreversible damage of the tertiary structure. Most enzymes are usually denatured by temperatures over

45 c .

Bacteria can grow productively in a wide variety of conditions, including, air, soil, water, and living organisms. (They are generally small, as they are prokaryotes – no ‘true’ nucleus). Their most favourable temperatures are 25-45 c, but some manage to survive in temperatures of 0 c and below- where they grow very slowly- whereas others are able to survive in above 80 (i.e. hot springs).

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45 c .

However, fungi are eukaryotes (they generally have larger cells, and membrane bound organelles), they also have a smaller range of habitat. They compromise mushrooms, moulds, yeast’s etc... They can be saprophytic, feeding on dead organic matter, or parasitic. Moulds which are multicellular fungi, grow best at about 30 c, their growth is slowed at lower temperatures.

A bacterium is heated to temperatures of 100 c or more in order to kill it, for example in food contamination. Whereas in order to kill off moulds, food is only heated to 60-70 , therefore showing that bacteria is much more tolerant to temperature.

Bacteria and fungi, similarly, both use amylases in their basic method of feeding, which plays an important role in their feeding digestion. Therefore, I propose that, due to bacteria’s larger range of habitat, that bacterial amylase will be more resistant to denaturisation at higher temperatures, than that of fungal amylase.

Apparatus

48 test tubes

5 water baths (40(control) 50, 60, 70,and 80 c )

30cm bacterial amylase solution

30cm fungal amylase solution

60cm starch solution

24 pipettes

5 Thermometers

Iodine solution (iodine dissolved in potassium iodide)

24 Spotting tiles

Plan

The presence of starch can be detected using a reddish/brown solution, made up of iodine dissolved in potassium iodide. From previous group experiments, I know that if starch is present, the solution should change colour to a dark blue/black. Therefore, the solutions will be denatured, when the amylase hydrolyses the starch to maltose, which should turn the iodine solution to an orange/yellow.

1.Measure 2cm bacterial amylase solution into a clean test tube,

2.Measure 2cm of 1 % starch solution into another clean test tube.

3.Place test tube containing enzyme solution into a water bath, of temperature exposure 50 c, and test tube containing starch solution in a water bath into a water bath of control 40 c, and leave for 10minutes - place a thermometer in the tubes, to ensure correct temperature is maintained.

4.Using a pipette, place a drop of iodine into each dimple in a dimple tray

5.At exactly 10minutes, remove the enzyme from temperature exposure, and leave in control water bath ( 40 c) for approx 1min – or until the enzyme has cooled to control temperature.

6. When enzyme has cooled to control, mix it with the substrate – starch solution.

7. After1 minute, using a pipette, spot one drop of the solution, into a pit containing one drop of iodine solution on the spotting tile, and record colour change.

8 .Repeat step 7 every minute until denaturisation occurs.

9 .Repeat steps 1-8, but substitute the temperatures of the water baths for; 60 c, 70 c,and 80 c respectively.

10. Repeat steps 1-9, but substitute bacterial amylase for fungal amylase.

11. Repeat steps 1-10 at least two times, an order to eliminate any anomalous results.

Variables

As a means to ensure that the test is fair, I shall only vary two factors (at different stages, not simultaneously.) I am going to research the effect of temperature exposure, on the rate of reaction of bacterial amylase, and fungal amylase.

I shall use carefully observed thermometer readings, as to ensure that the temperatures of my solutions are kept at a constant.

I shall also ensure that throughout my experiments, concentration of both bacterial and fungal amylase are kept precise and at a constant and exactly 2cm of each is used in each experiment.

I must also ensure that the concentration of my substrate- starch- is kept constant, at 1%, and I shall make it up of a buffer solution of ph7 –neutral – so as to eliminate any pH variations, which could effect the accuracy of my results and that again, the volume is kept accurate and constant, at 2cm .

It must also be taken into consideration, that denaturisation is time-dependant, so, prolonged exposure to a high temperature will have a greater affect than exposure for a brief time-lapse. Thus, when experimenting, I need not only consider the temperature at which each enzyme is treated, but I must also take careful precaution as to not exceed my timed exposures (10mins)

I must also ensure that the same size drop of iodine is used in each spotting tile pit, ( or as closely sized as I can approximate) and that the same concentration of iodine is used throughout.

Risk assessment

Starch solution: Very low risk, but has to be kept in a clearly labelled container.

Amylase enzymes: Low risk, although, all enzymes have biological activity, therefore need to

be treated with care, and handled carefully. Eye protection must be worn – as 0.15% solutions, and if in powdered form, inhaling must be avoided. Any spillages must be washed from skin as soon as possible.

Iodine solution: Can be toxic and irritant, therefore eye protection must be worn, and any

spillage’s washed from skin with water immediately. Small drops must be used, and tiles should be rinsed carefully under warm running water. Bottles must be clearly labelled.

Water Baths: Hands should, wherever possible be kept out of water/ice, especially at the

higher temperatures i.e. 80 c, as minor burns can be incurred. If burns incurred, skin should be put immediately under cold running water, and medical advice sought.

I must also ensure that any shards of broken glass are cleared away thoroughly, so as to eliminate unessessary injury, and that they are placed in a separate disposal unit to ordinary waste product.

I must also wear protective clothing at all times so that I do not either contaminate my solutions, or wreck my outer garments if spillages do occur, and I must wear safety goggles, in order to protect my eyes.

All solutions must be disposed of carefully – i.e down the sink (as the solutions that I will be working with are not highly toxic) and then the sink must be rinsed thoroughly.

All apparatus must also be washed and sterilised, as the majority will be used in future experiments – so as to avoid any possibility of future cross-contamination.

In the event of an outbreak of fire, all substances must be left inside the building, and caution must be taken to place them so that they are not at risk of being spilt, then sharp exit must be ordered through the nearest fire exit.

Organisation must also be taken into account, and ensured that all test tubes and containers are clearly labelled, and when finished stored correctly, so again to eliminate any spillages etc..

It must also be ensured that long hair is securely tied back, and if ties or loose clothing is worn, that they too are securely fixed.