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Background – Enzymes are proteins that operate as organic catalysts for biochemical reactions. They facilitate the reactions that occur in the body by lowering the activation energy required for reactions. One example of an enzyme is amylase, an enzyme that facilitates the hydrolysis of starch into its glucose monomers. In humans and other mammals, amylase is found within the saliva and pancreas, and amylase is also present within other living organisms such as plants and fungi.
Enzymes are affected by changes in temperature and pH, and all enzymes have specific ideal conditions in which an enzyme under those conditions will operate at optimum efficiency. Conversely, an enzyme outside of its ideal conditions may function less effectively in its task due to denaturation, the process where the protein structure of an enzyme irreversibly alters, rendering said enzyme less effective or completely useless. Therefore, the effect of a change in pH or temperature on an enzyme can be measured through the rate of reaction of that specific reaction, which can be measured in various ways.
The rate of a given reaction can be affected by the temperature, pH, concentration of reactants, and the enzymes present within the reaction. In order to measure the effect of changes in pH for an enzyme, in this instance amylase, temperature and concentration of both the substrate (the reactant for the enzyme, in this case starch) and the enzyme itself must be kept constant. By removing these other confounding factors, the relationship between pH and the efficacy of amylase can be accurately determined.
In the case of this experiment, an iodine/potassium iodide solution will be used as an indicator for the contents of starch within the solution. This solution signals the presence of starch in solution by allowing iodine ions to form organic complexes with starch molecules, creating a dark color, which dissipates with a decrease in starch concentration. Therefore, the effectiveness of amylase can be measured through a colorimeter, since as the concentration of starch within a sample decreases, the color of the solution will brighten and allow more light to pass through the colorimeter, a change that can be measured and used to determine the rate of a reaction.
Context of investigation – Enzymes have different ideal pH range while also losing effectiveness at different rates. This lab serves to determine the extent of that loss of effectiveness within a highly limited range for brewing amylase, used in the fermentation of malt liquor.
Ho: There will be no significant change in the effectiveness of amylase within a range of +-1 pH
Ha: Due to the sensitivity of the amylase enzyme, there will be a significant decrease in effectiveness across the 7+-1 pH range
The procedure is an adaptation of common amylase colorimetric assay procedures, where the concentration of amylase determines the absorbance of the sample. This origin of a procedure then took inspiration from high school lab experiments concerning measuring the activity of an enzyme under differing pHs. The setup and procedure were formed from a combination of these two inspirations, creating a lab procedure that essentially measures enzymatic activity through color change.
In order to set up this lab, stock solutions of 2mg/ml brewing amylase and 1M starch solution diluted 1:2 with distilled water were prepared. The amylase solution was intentionally created with a relatively high concentration in order to ensure that something could actually be measured, as previous trials with approximately 11mg amylase mathematically determined to react with a total of 1/8 moles of starch proved to be far too slow to be measurable in a reasonable timeframe. The original starch solution was diluted in order to both preserve starch and hopefully have little enough to react in a reasonable manner with an unusually high amylase concentration. In order to measure the effectiveness of the amylase consistently, 10 mL of starch would be drawn, followed by 1 drop of iodine/KI indicator; this would be the testing solution. In order to alter pH, separate starch solutions were created, with each starch solution titrated to a specific pH using buffer pills.
The procedure for the lab is rather simple. For the 4mL colorimeter cuvets, 2 mL would be filled with iodine-reacted starch, with 1.5 mL amylase solution being added to the cuvet immediately before beginning data collection. These cuvets would then have their light absorbance measured for five consecutive minutes, with data recorded at the initial start, and every minute afterward. Minute intervals were decided on so as to not make the data collection process too tedious while also allowing to show the potential curve created by the complete reaction of starch with amylase.
In order to preserve the validity of pH being the independent variable with light absorbance being its independent variable, several factors were kept constant, while others were ignored. The concentration of starch, amylase, and iodine were kept constant and their ratios kept consistent, as concentration is another major factor affecting enzymatic activity. However, temperature and light were disregarded as the experiment would occur under the exact same conditions for all trials, and enzymes are not affected by light. While enzymes are denatured by heat, the room was room temperature, not hot.
All data in the graphs shown are processed from the data collected and shown in Appendix A.
In comparison to the solutions without any enzyme, it can be seen that the solutions containing amylase follow a curved decrease, while the solution without any amylase follows a linear decrease. This shows a few important things. Firstly, this shows that the obtained amylase works, a worry when obtaining chemicals online. Also, this shows the correction required against the collected data, as the natural degradation of starch would work even with amylase in the solution.
At first glance, the data seems consistent; if one were to disregard the pH 7.5 outlier, one can observe the minute changes of rate of change across pH changes. However, a worrying statistic is the standard deviations of the rates of change, which, at times, end up higher than the original value. This implies a wide range of possible points, which questions the validity of collected data since anything that falls in that point could easily alter the actual results.
T-tests conducted on the data shows that the data is statistically significant, which is therefore most likely not due to chance, on pH ranges 6.5, 7, and 7.5. While this is important for confirmation as to the effectiveness of amylase at this pH, a p=0.62 at pH 6 and p=0.07 at pH 8 practically defeats the purpose of the entire lab, as it makes practically half the data pertaining to the sensitivity of amylase to pH changes invalid. Therefore, due to the uncertainty of the data, the lab fails to reject the null hypothesis.
The t-test showed p-values for all pHs, when compared to the ideal range of pH 7, to be statistically significant (p<0.05). However, the raw data itself tells a different, completely nonsensical, story. For some data sets, the standard deviation of the average rates of change were more than an amount equal to or greater than the original average value! Furthermore, by simply looking at the data one can see that, despite allegedly being statistically significant, the average rate of change for pH 7.5 makes no sense when considering the expected “bell curve” of enzyme efficacy in relation to pH. Furthermore, as previously mentioned, data sets for pH 6 and pH 8 were shown to be statistically insignificant with their t-tests. Therefore, the t-test only proved the statistical significance of completely insignificant data, leading to the rejection of the null hypothesis.
The sensitivity of the topic being researched opened up the possibility for a large number of errors. Firstly, the pH meters provided by the school were rather inaccurate, fluctuating between 0.3 pH when the experiment itself measures across differences of 0.5 pH levels. Furthermore, due to the dispersion of iodine in the starch solution, the initial absorbance for trials would not start close to the same range. This affected the averages produced, as a wide range of averages would affect how close the actual result is to the experimentally found result. Finally, the concentration of the amylase mix was unknown and undisclosed. It is possible that the amylase was mixed with an inert substance in order to bulk the amylase, thus making the concentration lower than it appears. All of these factors would lead to an inconclusive lab result, such as was observed here.
In order to more accurately determine the sensitivity to change of these chemicals to pH, some considerations have to be made. An obvious consideration is the use of more accurate equipment, namely pH meters, so as to ensure that the desired pH is reached. With more accurate pH meters would also be more accurate math done with regards to how to accurately create a solution with a specified pH, as the water in each solution of starch and amylase will affect the pH of the final solution. Perhaps both starch and amylase solutions should be titrated to the desired pH in order to ensure that when the two are mixed the desired pH is still reached. With these changes, perhaps an actual result can be found.
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