UNDERSTANDING THE EFFECT OF PH LEVELS ON ENZYME ACTIVITY

Christie Hilgert
christie.hilgert@my.unt.edu







Biology 1730.518
October 23, 2013








Aaron Harkleroad
Wednesday 8-10:50am










Abstract

This experiment examined the effect of different pH levels on the enzyme amylase.
Three different pH buffers were used to test how amylase reacts in different pH
levels. This was determined by adding drops of iodine at timed intervals to see if
amylase hydrolyzed starch at different pH levels. The results of this experiment
showed that amylase hydrolyzed the starch in the pH 7 buffer. However, amylase
did not hydrolyze starch in the pH 2 buffer or pH 12 buffer. This experiment showed
that amylase works best to hydrolyze starch at a pH level of 7.

Introduction

Hydrolysis is a process that disassembles polymers into monomers by breaking
them down with the addition of a water molecule. Amylase is an enzyme, a
specialized macromolecule that speeds up a chemical reaction (Reece et al. 2011).
This particular enzyme works in hydrolyzing starch, a glucose polymer from plants,
into smaller polysaccharides and the disaccharide maltose (Reece et al. 2011). When
amylase reacts with starch, it breaks apart the disaccharide maltose, which is two
glucose molecules linked together, through hydrolysis (Wang, n.d.). Iodine turns a
dark color when starch is present, but will stay a lighter color in the presence of
glucose. Three different pH buffers were used and the color of the iodine reacting in
the different concentrations was measured over time to determine which pH buffer
amylase hydrolyzed starch best in. For enzymes, there is a certain range at which
they work best in, and going outside of this range results in lower enzyme activity
(Worthington 2013). The hypothesis tested states that: Amylase will best hydrolyze
starch at a neutral pH level as opposed to extreme pH levels.

Materials and Methods

A spot plate with three rows (labeled A-C) and four columns (labeled 1-4) was filled
with five drops of starch and two drops of three different pH buffers (pH 2, pH 7,
and pH 12) and mixed thoroughly. As close in time as possible, one drop of amylase
was added to each well on the spot plate and stirred, as shown in Table 1.
Immediately following this, one drop of iodine was added to each of the wells in
Column 1 and stirred. This is considered Time 0, and observations on the effect of
color from the iodine were recorded. One drop of iodine was added to the next
column and stirred every 5 minutes, and observations were recorded for a total of
15 minutes.
Preparation of Spot Plate
Column 1
Time 0 min
Column 2
Time 5 min
Column 3
Time 10 min
Column 4
Time 15 min
Row A: pH 2 5 drops starch
2 drops pH 2
buffer
1 drop amylase
5 drops starch
2 drops pH 2
buffer
1 drop amylase
5 drops starch
2 drops pH 2
buffer
1 drop amylase
5 drops starch
2 drops pH 2
buffer
1 drop amylase
Row B: pH 7 5 drops starch
2 drops pH 7
buffer
1 drop amylase
5 drops starch
2 drops pH 7
buffer
1 drop amylase
5 drops starch
2 drops pH 7
buffer
1 drop amylase
5 drops starch
2 drops pH 7
buffer
1 drop amylase
Row C: pH 12 5 drops starch
2 drops pH 12
buffer
1 drop amylase
5 drops starch
2 drops pH 12
buffer
1 drop amylase
5 drops starch
2 drops pH 12
buffer
1 drop amylase
5 drops starch
2 drops pH 12
buffer
1 drop amylase

Table 1: volume and concentration of starch and pH buffer used to prepare spot plate
Results

Amylase hydrolyzes starch at different efficiencies depending on the pH level. Iodine
stains starch dark, but does not stain glucose. The wells in Row A had the pH 2
buffer, which has an acidity condition similar to that in the stomach. The wells in
Row B had the pH 12 buffer, which is very basic. In both of these solutions, amylase
did not hydrolyze starch, as these conditions are nowhere near the enzymes natural
environment, and both rows stained dark in the presence of iodine. The wells in
Row B had the pH 7 buffer, a neutral pH level, and hydrolyzed starch more
efficiently as the neutral condition is closest to the enzymes natural environment.
Row B did not stain dark in the presence of iodine. In Time 5 of Row A, the color was
abnormal and lighter than expected, but the proceeding experiments data was
conclusive with the expected results. In Time 0 of Row B, the amylase did not have
time to break down the starch, proving why the coloration was dark instead of the
expected light. These results support the hypothesis that amylase works best in a pH
level of 7, as iodine did not stain the wells of Row B, but did in the wells of Row A
and Row C. The results from this experiment are shown in Table 2.
Results of Enzyme pH Activity
Buffer Column 1
Time 0
Column 2
Time 5
Column 3
Time 10
Column 4
Time 15
Row A: pH 2 Stained dark
Starch present
Unstained
Glucose
present
Stained dark
Starch present
Stained dark
Starch present
Row B: pH 7 Stained dark
Starch present
Unstained
Glucose
present
Unstained
Glucose
present
Unstained
Glucose
present
Row C: pH 12 Stained dark
Starch present
Stained dark
Starch present
Stained dark
Starch present
Stained dark
Starch present
Table 2: Iodine coloration in spot wells showing the effect of pH on amylase effectiveness
Discussion

Due to the lack of dark coloration in the pH level of 7, it can be concluded that the
hypothesis was correct. The enzyme amylase works best in hydrolyzing starch at a
neutral pH of 7 as opposed to extreme pH levels. The pH of 7 is nearest to the
natural environment of amylase, whereas the activity of the enzyme is lowered due
to the denaturing of the enzyme when the pH levels reach extreme ends. These
experimental results are conclusive with the expected results in the hypothesis, and
test adequately for the presence of starch in different pH levels over time.















References

Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Jackson, R. B.
(2011). Campbell Biology (9
th
ed.). San Francisco, CA: Pearson
Benjamin Cummings.

Wang, N. S. (n.d.). Starch Hydrolysis by Amylase. Retrieved from
http://www.eng.umd.edu/~nsw/ench485/lab5.htm

Worthington Biochemical Corporation (2013). Introduction to Enzymes. Retrieved
from http://www.worthington-biochem.com/introbiochem/effectsph.html