Subtle Bacillus
The bacteria can be extracted from the digestive system of a dead ruminant. The bacterium is highly flagellated and may therefore travel through liquids in the digestive tract. Additionally, research indicates that the bacterium is quite receptive to genetic engineering. Ruminant guts have more concentration because they have holes that are vulnerable to food contamination. The bacterium taken from a ruminant's gut will provide the enzyme amylase (Rajagopalan & Krishnan, 2008).
Starch
Potatoes will be used as the source of the starch.
Type of treatment to use:
Type of solutions: 2.0ml amylase solution, 2.0ml starch solution, and 5.0ml pH buffer solution (a mixture of weak acid and conjugate base).
pH of the solutions: pH of 6.0, 7.0, and 9.0. The pH of above 7 will be obtained from the triethanolamine buffer, while ammonia buffers can be used to get the pH of less than 7, because they are not highly stable.
3 drops of Iodine solution into each test-tube.
Length of exposure: 60 seconds.
The enzyme activity is determined by observing the rate of starch breakdown, or formation of maltose.
Samples treatment:
pH treatment at 6.0, 7.0, and 9.0.
Control in the experiment: Boiled Amylase. Just like the other enzymes, amylase is a protein, and therefore, boiling affects its molecular structure.
Sample size:
3 sample sizes.
Conducting Experiment
Single drop of solution of iodine is placed on a tile lined in rows.
Test-tubes are labeled with a pH of 6.0, 7.0., and 9.0.
2.0ml of the amylase solution is added into each test-tube with a syringe.
5.0ml of a solution of buffer is added into each test-tube.
With another syringe, 2.0ml solution of starch is added the amylase solution and mixed using plastic pipette.
After 10.0 seconds, plastic pipette is used to put a drop of the mixture on each drops of the iodine solutions. The solutions of iodine should change to blue-black to show the presence of starch.
Repeat by adding another drop of the mixture into the next drop of the iodine solution.
The procedure is repeated until the solution of the iodine and the mixture of the amylase buffer and starch remain orange in color. The drops of iodine solution should be added all the starch substrate has been dissolved, leaving behind the amylase enzyme. The orange color is an evidence of amylase.
The whole procedure is repeated with different pH buffers and data collected.
A graph is the plotted for the starch to be broken down against the pH.
Reference
Rajagopalan, G., & Krishnan, C. (2008). α-Amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate. Bioresource technology, 99(8), 3044-3050.
