The independent variable that I will be changing for this experiment will be ‘the temperature at which the enzyme-substrate reaction takes place’. Therefore all other variables must be kept constant. In order to do this the following must be done:
The amount of enzyme (amylase) used: To keep this constant, in each amylase and sodium alginate mix, the same amount of amylase will be used (4cm ).
The amount of sodium alginate used: To keep this constant, in each amylase and sodium alginate mix, the same amount of sodium alginate will be used (6cm ).
The amount of substrate (starch solution) used: To keep this constant, I will use the same amount of starch solution in each beaker of 50cm that I use.
The concentration of the starch solution: To keep this constant, I shall use the same starch solution in each 50cm beaker full that I use.
The concentration of amylase solution used: To keep this constant, I shall use the same amylase solution in each 4cm syringe uptake that I do.
The concentration of sodium alginate solution used: To keep this constant, I shall use the same sodium alginate solution in each 6cm syringe uptake that I do.
The dependant variable that I will measure will be the time it takes for all the starch to be broken down.
To get a set of quantative results, the time at which the iodine goes deep brown will be measured, in both free and immobilised enzymes, against the temperature of the water bath.
Method
The following method will be used to perform the experiment:
1. Collect all equipment.
- Measure up 50cm of starch solution into each of two clean beakers clearly labelled.
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Place the two beakers containing the starch solution into a water bath at 30°C.
- Measure up 50cm of calcium chloride solution into each of two clean beakers, clearly labelled.
- Draw up 4cm of amylase and 6cm of sodium alginate into a 10cm syringe.
- Turn syringe upside down 10 times to mix the contents.
- Gently release the contents of the syringe, drop by drop, into a beaker containing calcium chloride . Swirl the contents of the beaker whilst doing this, to ensure equal sized beads.
- Filter the contents of the beaker, in a funnel with filter paper, and then rinse the beads with distilled water for 60secs.
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Place beads in a boiling tube and put in a water bath at 30°C
- Draw up 4cm of amylase and 6cm of distilled water into a clean syringe.
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Place this amylase-water solution in a boiling tube and place in a water bath at 30°C.
- Empty the contents of both boiling tubes (at the same time), separately, into the two beakers of starch solution.
Start the timer at this point.
- At intervals of 30seconds, test the contents of each beaker for starch by removing small, equal amounts with droppers and adding them to a few small drops of iodine on the spotting tiles. The droppers must be rinsed with distilled water after each test.
Repeat the above process using water baths at different temperatures: 40°C, 50°C, 60°C and 70°C.
If starch is still present the iodine will go black/blue. If starch is not present the iodine will go deep brown.
To ensure my results are reliable I will do the experiment 3 times. To ensure I work safely I shall wear protective goggles and I shall behave according to GLP (General Laboratory Practice.
To make the experiment a fair test, I shall:
- Replace the sodium alginate with distilled water in the free enzyme solution.
- Use equal amounts of starch in each beaker.
- Make sure to the best of my ability that all beads have the same surface area.
- Make sure all the 30second timed intervals are exact.
- Make sure the same amount of iodine is used in each trough on the spotting tile.
- Make sure the same amount of enzyme is used in both free and immobilised mixes.
- Make sure the end point reached in the iodine is the same each time.
