“Effects of Amylase reaction time when breaking down starch.”
Experiment Goal: Our goal was to study the time it takes for the amylase enzyme in saliva to decompose starch and understand how digestion works.
Hypothesis: We expect that a higher concentration of the amylase enzyme in our saliva will lead to a faster breakdown of starch.
Materials: A brightly colored test tube rack, kim wipes, a trash beaker, and 1.5 ml microcentrifuge tubes.
Test plate Micropipetters and tips DI water Buffer solution … 4. 5 to 8. 8 I2Kl (grams iodine) Starch solution Enzyme (amylase) 80 degree Celsius water (HOT) Floating test rack
Procedure: While controlling the amount of starch and the amount of buffer we use with a pH of 5. 8, we want to investigate how changes in enzyme concentration affect reaction rates. First we put 500 ml of amylase from 0. 2% solution (provided by Michael Bunch) into an experimental tube. We then put 3 drops of Iodine into a test plate (the color is brown).
Upon adding 250 ml of starch solution to the buffer-amylase mixture, the reaction commenced promptly. Initially, we timed the reaction after 20 seconds, during which we extracted a 25 ml sample of the mixture and introduced it into an iodine test site. Instantly, the brown iodine transformed into a purple hue. The presence of purple traces in the iodine-amylase mixture suggests that the amylase was unable to fully decompose the starch due to insufficient time. We repeated this procedure every 20 seconds for a duration of three minutes.
The amylase’s ability to break down starch was initially ineffective, prompting us to increase the concentration of the amylase solution to 0.5% and restart the experiment. After reestablishing the experiment setup, we prepared a mixture of amylase and starch and began timing intervals at 10 seconds. Within this short time frame, the iodine turned brown, suggesting that increasing the amylase concentration facilitated a faster reaction without being excessively rapid. However, because the reaction became too fast for thorough observation, we diluted the 0.5% solution to slow down the reaction time.
We attempted to use a 200/800 enzyme to buffer or 0.1% of the 0.5% solution. After combining the solution and observing the timing again, we determined that 10 seconds remained too rapid and the iodine still appeared brown. Considering another diluting solution, we diluted the 0.5% dilution to a 100/900 ml solution, yet even after 10 seconds it was STILL TOO FAST. We then attempted a 60/940 ml solution, but unsurprisingly, the reaction remained too rapid. As our options dwindled, we ultimately decided to try one final solution of a 20/980 ml and discovered that after 10 seconds, the iodine finally turned purple.
We timed the reaction at intervals of 10 seconds. After 20 seconds, we took another 25 ml sample. At this point, the starch had dissolved and the iodine turned brown. We experimented with different enzyme to buffer ratios to slow down the reaction time. We discovered that a 20/980 or 1/50 ratio resulted in the amylase breaking down the starch in 20 seconds. Intrigued, we also tried a 10/490 solution. We found that after 10 seconds, the mixture turned purple. After 20 seconds, it remained purple, and at 30 seconds, the starch had dissolved and the iodine turned brown.
Conclusion: During this portion of the experiment, it was determined that our hypothesis was accurate. We discovered that concentration played a vital role and required significant dilution in order to observe the effect in slow motion. Moving on to PART II of the enzyme vs starch experiment, the objective was to determine if the pH of the buffer impacted the reaction time.
Hypothesis: Having learned that amylase concentration is a significant variable, we predict that pH will have the same effect. Specifically, we anticipate that a lower pH will result in a faster reaction time.
Procedure: We conducted the experiment using the same setup and design, controlling the solution by excluding certain components and using a 10/990 enzyme to buffer solution. However, we varied the pH of the buffer. Initially, we set the pH level to 4.5 and began mixing and timing the reaction in 5-second intervals. The starch remained for a duration of 20 seconds. Upon discovering that acidic pH levels resulted in a quicker reaction time, we decided to test a base pH level buffer of 8.8. We followed the same procedure as before, using a test tube containing 490 ml of buffer, adding 10 ml of amylase solution, and finally adding 250 ml of starch.
We began by timing the starch breakdown in 10-second intervals. The reaction time exceeded 4 minutes, so we terminated this round of tests. We still had one more pH level to examine, and our hypothesis seemed accurate. For a pH level of 7.4, we observed that the starch completely broke down within 4 minutes using 20-second intervals and the same solution. Therefore, in part II, we concluded that our hypothesis was confirmed: as the pH becomes more acidic, amylase can break down starch at a faster rate.
Part III of the Amylase vs Starch experiment aimed to investigate the impact of temperature on the reaction time, taking into account the understanding gained about amylase, starch, and pH level reactions. The hypothesis proposed that as water temperature increased, the reaction time for starch breakdown would decrease. The procedure involved using 990 ml of a pH 7.4 buffer, 10 ml of enzyme, and 250 ml of starch situated in a floating test rack within an 80-degree Celsius water bath. The experiment required a two-minute heating period before the mixing and timing of solutions could commence.
After heating the elements, we blended the solution and began timing at 10-second intervals. It was observed that once iodine was added to starch, the reaction produced a vibrant purple color. We continued timing for 4 minutes and noted that the starch remained intact. Our conclusion is that during part III of the experiment, our hypothesis was proven incorrect as the reaction had an opposite effect. The enzymes’ ability to break down starch is significantly hindered by heat. With such a short duration, we were unable to achieve starch breakdown. It is evident that the amylase enzyme functions more rapidly in a cooler environment.
