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Optimal Temperature and pH for the Reaction of α-Amylase and Starch
Abstract
Enzymes are used in living organisms to make vital chemical reactions more energy
efficient. These enzymes are most effective at specific temperature and pH ranges. In this
experiment, various temperature and pH levels are tested to find the optimal environment for the
hydrolysis of starch by α-amylase. When starch is introduced to I2KI, the solution turns a
bluish-purple color. The absorbency rating of this colored solution can be used to determine how
much starch is left in the sample. After conduction tests at five different temperatures and six
different pH levels, the data shows that the optimal environment for the starch/amylase reaction
is at 64°C in a solution of pH 4.5.
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Introduction
Chemical reactions are important to all living organisms. Within the organism, proteins
called enzymes facilitate the reactions. Enzymes cause the activation energy for the reaction to
be lowered, accelerating the reaction and making it more efficient. Veit (1996) states that the
enzymes are not consumed or permanently changed in the chemical reaction. He also goes on to
state that an enzyme cannot cause inefficient reaction to occur.
All chemical reactions occur at an optimal temperature and pH level. By increasing the
heat in the system, the chemical reaction increases, but only to a point. Also if the pH is to
acidic or basic, the reaction will either not occur or be inefficient. In order to find these optimal
levels, I studied the effects of temperature and pH on the rate of reaction of α-amylase with
starch.
Amylase is an enzyme that is harvested from germinating barley seeds. This enzyme
hydrolyzes starch molecules into glucose that the seedling can use. Torchia (1999) found that
starch could be hydrolyzed at low temperatures with the help of α-amylase. His study showed
that with the help of α-amylase, starch could be hydrolyzed at temperatures where the reaction
would not normally occur. Ibrahim et al. (2004) varied the pH of the solution in order to find the
optimal condition for starch hydrolysis. They found a pH of 7 was optimal for their solution
mixture and application. The optimal ranges of pH and temperature could change depending on
substrate concentration, the amount of enzyme present, and the other molecules present in the
solution.
I hypothesized that at higher temperatures the reaction rate would continue to increase. I
also hypothesized that the optimal pH for the reaction was closer to a neutral level.
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Materials and Methods
The experiment was conducted on June 1, 2004 in Carr Hall room B-14 at the University
of Florida. Five temperature tests and six pH tests were conducted. The temperatures selected
for this study were 15 °C, 35 °C, 45 °C, 50 °C, and 64 °C and the pH levels were 4, 4.5, 5, 5.5, 6,
and 6.5.
I prepared a reaction flask containing 35 ml of the stock starch solution and 35 ml of
distilled water for each test. In the case of the pH tests, the distilled water was buffered to the
appropriate pH level before being added to the starch solution. Twelve test tubes were also
prepared for each test. A “blank” for the spectrophotomer was created by placing 5 ml of
distilled water (buffered in pH tests) and 0.1 ml of I2KI in a test tube. The rest of the empty tests
tubes were prepared by adding 0.1 ml of I2KI.
I heated up the reaction flasks to their specified temperatures before adding any enzyme.
Five milliliters of the solution was removed from the flask and added to one of the test tubes to
determine the solution’s initial absorbency. Upon determining this initial reading, I added 1 ml
of the α-amylase solution to the reaction flask and started a timer. I took 5 ml samples every
minute for the first eight minutes then at 10 minutes and 20 minutes for a total of 10 samples.
The absorbency values were recorded for each sample.
The pH test followed the same basic procedure as the temperature test. I took an initial
sample then added the enzyme. Samples were taken every two minutes for 20 minutes. The
absorbency values were also recorded in a table for each sample.
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Results
The absorbency values for the temperature tests are displayed on Fig. 1. The absorbency
values for the pH tests are shown in Fig. 2. The higher the absorbency value, the more starch
remains in the sample. In all the tests, I observed a decrease in the amount of starch as time
progressed.
With the data obtained from the temperature and pH tests, I was able to calculate the
reaction rates. The calculations needed to determine reaction rates are displayed in Table 1 for
the temperature tests and Table 2 for the pH tests.
Figure 3 shows the reaction rates plotted against the various temperatures. This figure
shows that the optimal temperature for the hydrolysis of starch in the presence of α-amylase is
64°C. Figure 4 plots the reaction rate versus the pH level of the solution. In this figure, it is
apparent that the optimal pH level for the reaction is 4.5.
Discussion
From the data gathered it is apparent that as you increase the temperature of the solution,
the reaction rate accelerates. While this relationship could continue on an upward trend, it is
possible that, like the pH tests, the reaction will reach an optimal temperature before the reaction
rate starts to decrease. This value does not appear to be in the temperature range selected for the
experiment. With the data available, the temperature tests validate my hypothesis.
After calculating the graphing the reaction rates for the pH test, it appears that this
reaction prefers a moderately acidic environment. The reaction rate peaked at a pH level of 4.5
which does not follow with my original hypothesis. The data shows that a small amount of extra
hydrogen ions helps the reaction accelerate.
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From all the data gathered, I would say that in order to fully optimize this reaction, it
should be preformed at 64°C in a solution with a pH level of 4.5. In this optimal environment, αamylase is working at peak efficiency to breakdown starch into usable glucose.
If I was to perform this experiment over again, I would concentrate on a higher
temperature range in order to determine where the temperature peaks for this reaction.
Literature Cited
Ibrahim, N. A., M. El-Hossamy, M. S. Morsy, B. M. Eid. 2004. Optimization and Modification
of Enzymatic Desizing of Starch-Size. Polymer-Plastics Technology & Engineering. 43:519-539
Torchia, Mark G. 1999. Enzymes can be used to hydrolyze starch at low temperatures.
Chemtech. 29:3-9
Vliet, K.A. (ed.). 1996. A Laboratory Manual for Integrated Principles of Biology: Part One –
BSC 2010L. Ginn Press, Needham Heights, Massachusetts.
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