Effects Observed on Enzyme Activity Caused by Variable Enzyme Concentration, pH, and

Temperature
Kimberly Williams, Kerry Roszell, Faris Al Bakhat, and Andrew VanDam
Biology Lab 213 section 000, George Mason University, Virginia
17 November 2013

Introduction
In a technologically advanced society it is known, all too well, the idea of being able to make
tasks easier by speeding them up. Computer technology has progressed toward goals of making
everything available at ones fingertips by speeding up many processes. Suppose it was necessary to
contact a friend who was in another country, before the advancement of our technologically saturated
world, a messenger was necessary to travel to that person and deliver it. This process spanned days or
even months and there were no guarantees it would arrive. Technology has taken this task and created
a reality that seemed impossible in a time not long distant.
Enzymes, similar to the analogy above, speed up processes in a cell. It catalyzes, or speeds up,
reactions that would otherwise take too long to occur for the cell to survive. Enzymes are specialized
protein structures that reduce the rate of a reaction without becoming altered or used up in the
reaction. Enzymes accomplish this by effectively decreasing the activation energy of a reaction.
Activation energy is the energy required to force a reaction or reactants from its stable state to an
unstable state, from which most reactions occur. Think of a ball on a hill. The ball sitting at the top of
the hill is in an unstable state, where it has taken energy to push it up the hill. The reaction will occur
as the ball rolls down the hill or as the ball reaches the top of the hill. By lowering the amount of
energy it takes to roll the ball up the hill, logically the entire reaction will happen faster. (Sadava,
151)
Another reason to understand how enzymes function is they play an important role in our own
digestion as humans. In a study at Cambridge, an experiment was performed on how factors affect
amylases ability to convert starch into energy. The hypothesis for the experiment was to understand
how to gain the most amount of energy from what is consumed by an individual. As amylase is better

understood, the information that was collected could be used to improve individuals health. (Tester;
Karkalas; Qi, 186)
The effects of enzymes can be witnessed through experimentation because specific enzymes
catalyze reactions in different ways. Experiments call for certain proteins depending on the
observations that are desired to be studied. The experiment that was used was visibly seen by the
color change in a solution; representative of chemical structures being broken down by catalysis.
Amylase is an enzyme commonly found in human saliva and germinating seeds. Amylase
breaks down starch into simple sugars. The end result is glucose, this sugar molecule fuels cell
activity in nearly every living organism. To perform the experiment Amylase was extracted from
germinating seeds. Two other items are necessary, a starch solution and a detecting reagent. Lugols
Iodine is the detecting reagent used to signify the presence of starch. A blue-black color indicates
starch is present in the solution. When Amylase is added to the solution containing starch and Lugols
Iodine, Amylase catalyzes the starch molecules. As the enzyme is added a dark blue-black color will
be present which will fade to colorless because of the absence of starch and Lugols Iodine only
reacts with starch. If the enzyme is within its optimal environment, the hypothesis is, the reaction
will proceed all the way through, leaving no blue color in the solution. (Fox Madden, pg. 63-64)
It is important to note that enzymes have certain environmental factors that need to be met for
optimal performance. Extreme temperature, pH, and harsh chemicals can unravel or denature protein
structures of an enzyme which renders it ineffective. Even with futile enzymes chemical reactions
occur, but at much slower rates. Knowing this truth, another hypothesis was proposed. The blue-black
color from the Lugols Iodine would still fade, but at a decreased rate, as the enzymes become
ineffective due to adjusted environmental factors. A colorimeter will be used to gather data on this
proposal by passing a beam through the contents of a test tube to obtain an absorption value for the
specific color it possesses (Fox, Madden, pg. 70).

The hypothesis presented was compared to the data gathered from a number of experiments
that were performed in order to evaluate how amylase reacts to varying environments. To prove the
first hypothesis positive a test will be performed that will create an ideal environment for amylase. It
is expected when starch, the substrate, is added to the solution; amylase will catalyze the substrate to
completion. This result will be visually observable by the absence of the dark blue tint from the
Lugols iodine and will appear yellow. If this occurs, it will be evidence that the data supports the
hypothesis. In addition to observing color change, the reaction time will be quantified in order to
understand how quickly this reaction is occurring, which will be used to compare with the second
hypothesis. In order to test the second hypothesis the environment will be changed by varying the
concentration of amylase by dilution, pH and temperature. As the environment is changed it is
predicted that the rate of production product will decrease compared to the rate of the enzyme in an
ideal environment. If it is observed that the time measured increases or does not go to completion
compared to the amylase in an ideal environment, this will be evidence to support the second
proposed hypothesis.
Materials and Methods (Variable Enzyme Concentration)
Enzyme concentration was adjusted to understand its effect on a constant concentration of
substrate. To vary the concentration of amylase a serial dilution was performed by measuring 30 mLs
of active amylase into a flask. Then 10 mLs was pipetted out and transferred into a flask containing
20 mLs of water. After mixing, the previous step was repeated for the third flask, resulting in three
different concentrations: undiluted, 1:3, and 1:9. Each flask should contain equal volumes of solution.
The spectrophotometer (set at 540nm) was zeroed after a blank cuvette containing 1.5 mLs of
water and 3 drops of Lugols iodine was prepared. Immediately after starch was added the time was
noted and the solution was mixed. 3 ml of solution was removed and added to a test tube containing 3
drops iodine, mixed, poured into cuvette and absorbance recorded promptly from spectrophotometer.
The previous step was repeated on 30 second intervals until 3 minutes was reached and thereafter on

1 minute intervals (approximately 13 minutes). For the remaining two flasks the process was repeated
as described above, including the spectrophotometer being zeroed out for the specific flask being
used.
After the results were graphed the effect of varying the concentration of amylase became
clear. In Figure 1 the raw data is shown from the experiment. To calculate the percent of amylose
present for a specific absorbance, which was recorded from the experiment, a standard curve was
used. The data in Figure 2 represents the absorbance converted to the correlating percent amylose.
From Figure 2 the graph in Figure 3 was assembled. Referencing the graph from Figure 3, a
qualitative comparison gives evidence that none of the concentrations have similar slopes when
comparing percent amylose to time. This dissimilarity shows that changing the concentration of an
enzyme clearly influences the enzyme activity with the substrate. The graph suggests that as the
concentration of the enzyme is increased the presence of substrate will decrease faster when
compared to weaker concentrations of enzyme. This trend holds true as well for decreasing the
concentration of the enzyme, meaning the presence of substrate will decrease more slowly.
A noticeable error occurred in the weakest concentration, to get a comparable value the
starting value was comparted to the ending value which gave an estimated slope. Aside from the fact
the value was estimated it is clear that the slope for the weakest concentration is significantly less
steep than the other two lines of higher enzyme concentration which supports the results discussed
above.
Materials and Methods (Variable pH)
During this phase the effects that pH and temperature had on enzyme reactions were observed.
A spectrophotometer was needed set at 540nm, water, Lugols iodine, four flasks, cuvettes, test tubes,
and a vortexer were needed.
To test the effects of pH, four flasks were used with varying concentrations. The

spectrophotometer was blanked after 3 mLs of water was mixed with three drops of iodine in a
cuvette. 22mLs of pH 4, 5, 6, and 7 was added to the four flasks, respectively. In flask one 2 mLs of
substrate (starch) solution and 1 mL of enzyme extract was added to the flask then mixed with a
vortexer. Immediately after 3 mLs of solution was transferred to a test tube containing 3 drops of
iodine. The test tube was vortexed and transferred to a cuvette. Once the cuvette was placed in the
spectrophotometer the absorbance was recorded. This process was repeated once every minute for
approximately seven minutes. For each the remaining flasks this process was repeated.
After the data in Figure 6 was evaluated, it was determined that the more acidic the solution
was the less enzyme activity was observed. It is evident from the data gathered from this experiment
as the acidity approached the level of that found in the human body, a pH of 7, enzymatic activity
increased.
Materials and Methods (Variable Temperature)
To test the effect of temperature on enzyme activity four flasks were used filled with 22 ml of
water and 2 mLs of starch solution then mixed. 1 mL of enzyme extract was added to each of the four
test tubes. Flask and test tube together were placed into an ice water bath at 4 degrees Celsius, for ten
minutes. Flask 2 and test tube 2 were tested at room temperature, 22 degrees Celsius. The content of
the test tube was added to flask 2 and swirled till mixed. The data was gathered by immediately
taking a 3 mL sample and added it to a test tube containing three drops of Lugols iodine. After
vortexed the solution was put into a cuvette and placed in the spectrophotometer and the absorbance
was recorded. This process was repeated every minute for seven minutes. After removing flask one
from the ice bath, the 1 ml of enzyme extract was added, mixed, and placed back into the ice bath.
Next a sample was immediately taken by extracting 3 mLs of solution and added to a test tube that
contained three drops of Lugols iodine. The solution was vortexed and poured into a cuvette and
placed in the spectrophotometer where the absorbance was determined. This procedure was
completed every minute for the next seven minutes. Results were then recorded in the appropriate

table. Flask three and four were incubation at 37 degrees Celsius and 70 degrees Celsius,
respectively, the same procedure was performed to take samples on set intervals and data recorded.
From the observations recorded in Figure 9, it was seen that as the temperature was lowered
below normal human body temperature enzyme activity slowed. It was also seen enzyme activity was
inactive in the 70 degrees Celsius flask. This was most likely due to the broken down tertiary
structure of the enzyme.
Conclusion
The point of the variable enzyme concentration experiment was to see how concentration of
enzyme would affect enzyme activity. The absorbance read from the spectrophotometer measured
how much starch was still present at a specific time after enzyme activity began. It was seen in the
table Figure 2 that the percentage for the undiluted and 1:3 enzyme dilutions decreased over time,
which was expected to happen. The undiluted enzyme concentration decreased at the highest rate,
whereas the 1:3 dilution decreased most gradually. This decreased rate of enzyme activity could be
attributed to the enzyme saturation theory, which implies that reaction rate slows as more enzymes
are used up and all active sites are occupied. In the 1:9 dilution in Figure 2, it was seen that the
percent of starch appeared to move in a wave instead of a steady downward curve, which can be seen
in Figure 3. This error was caused when the solution was extracted at the incorrect time from the flask
and put into the cuvette. While waiting to record the intervals the solution reacted inside the cuvette
instead of the flask which did not give an accurate account of the percent of starch for each specific
time.
The variable pH experiment was conducted to show the difference in enzyme activity when
changing the pH of the solution in which the reaction is occurring. The absorbance, like in the
previous experiment was read by the spectrophotometer to show the percent of starch still remaining
over time. Unlike the previous experiment the time only went to seven minutes instead of eight. As

seen in Figure 5, the percent of starch decreased as time went on for each experiment. The pH 4
solution started off with a higher percent of starch, 0.019, and did not lower beyond 0.019. This
shows that there was little to no activity that happened when amylase was exposed to a slightly acidic
environment. When the pH was increased to 5 more enzymatic activity was observed. The most
activity was seen when the pH was between 6 and 7. Although activity decreases slightly when the
pH was increased to 7, both exhibit increased enzyme activity in relation to pH 4 and 5. This
solidifies the theory that this particular amylase is specific to the human species because human saliva
operates most efficiently at a pH range of 5.6 to 7.9.
The effect of enzyme activity due to temperature shared a similar trend with pH. Recalling the
procedure for the variable pH experiment, four different temperatures were observed and absorbance
measured for the percentage of starch still present in the solution. The temperatures were 70, 37, 22,
and 4 degrees Celsius. Figure 8 represents the percent starch for each solution. At 70 degrees Celsius
the least amount of activity was observed, which can also be seen in the graph in Figure 9. At this
high temperature activity ceases. This was most likely due to the denaturing of the enzyme. At 37
degrees Celsius, the average temperature of the human body, the most activity was observed. This
observation was expected. During the experiment, as seen in Figure 7 but not easily seen in the graph
or table Figures 9 and 8, respectively, is a blank section for the fourth minute for the absorbance at 37
degrees Celsius. This was due to a wasted cuvette. Even though this reading was lost it was not
significant enough to distort the readings or interrupt the results for the other readings at 37 degrees
Celsius. There was little enzymatic activity at 4 degrees Celsius due to a decrease in collisions of
molecules because of a lower kinetic energy in the solution as the temperature was decreased. The
flask in this experiment was taken out of the ice bath while extracting the solution for readings, which
explains the consistent decrease in percentage of starch. As the solution warmed activity was able to
occur. If the solution was kept at 4 degrees Celsius enzymatic activity would have possibly mirrored
the activity at 70 degrees Celsius.

Appendix
(Figure 1)
Absorbance vs. Time Table
Time (min)

Undiluted Enzyme

1:3 Enzyme Dilution

1:9 Enzyme Dilution

2.661

2.903

2.83

0.5

2.383

2.808

2.528

2.138

2.663

2.865

1.5

1.84

2.489

2.743

1.559

2.194

2.317

2.5

1.193

1.99

2.38

0.91

1.833

2.734

0.68

1.774

2.727

0.479

1.693

2.647

0.358

1.608

2.582

0.294

1.534

2.554

1.529

2.017

(Figure 2)
Amylose % vs. Time Table
Time (min)
0

Undiluted Enzyme
0.042

1:3 Enzyme Dilution

0.046

1:9 Enzyme Dilution

0.045

0.5

0.038

0.045

0.040

0.034

0.042

0.046

1.5

0.029

0.040

0.044

0.025

0.035

0.037

2.5

0.019

0.032

0.038

0.015

0.029

0.044

0.011

0.028

0.043

0.008

0.027

0.042

0.006

0.026

0.041

0.005

0.024

0.041

0.000

0.024

0.032

(Figure 3)

Effects of Altering Enzyme Concentration

0.050
0.045
0.040
0.035
0.030
% Amylose 0.025
0.020
0.015
0.010
0.005
0.000

Undiluted Enzyme
1:3 Enzyme Dilution
1:9 Enzyme Dilution

0.5

1.5

2.5

Time (min)

(Figure 4)
Absorbance vs. time
Time (min.)

pH 4

pH 5

pH 6

pH 7

1.219

1.213

1.128

0.964

1.222

1.091

0.788

0.587

1.218

0.969

0.465

0.218

1.21

0.839

0.235

0.091

1.205

0.774

0.122

0.069

1.197

0.669

0.098

0.059

1.184

0.669

0.057

0.041

1.187

0.662

0.029

0.052

(Figure 5)
% Amylose vs. time
Time (min.)

pH 4

pH 5

pH 6

pH 7

0.019471

0.019376

0.018027

0.015425

0.020

0.017

0.013

0.009

0.019

0.016

0.008

0.004

0.019

0.013

0.004

0.002

0.019

0.012

0.002

0.001

0.019

0.011

0.002

0.001

0.019

0.011

0.001

0.001

0.019

0.011

0.001

0.001

(Figure 6)

Effect of Variable pH on Enzyme Activity

0.025
0.02
pH 4

0.015
% Amylose

pH 5
0.01

pH 6
pH 7

0.005
0
0

Time (min.)

(Figure 7)
Absorbance vs. time
Time (min.)

4 degrees C

22 degrees C

37 degrees C

70 degrees C

1.599

1.311

1.008

1.342

1.477

1.259

0.153

1.297

1.43

1.136

0.054

1.295

1.373

1.101

0.024

1.35

1.333

1.022

1.237

0.966

0.064

1.307

1.16

0.915

0.022

1.303

1.075

0.867

0.031

1.261

1.286

(Figure 8)
% Amylose vs. time
Time (min.)

4 degrees C

22 degrees C

37 degrees C

70 degrees C

0.026

0.021

0.016

0.021

0.024

0.020

0.003

0.021

0.023

0.018

0.001

0.021

0.022

0.018

0.001

0.022

0.021

0.016

0.000

0.021

0.020

0.015

0.001

0.021

0.019

0.015

0.000

0.021

0.017

0.014

0.001

0.020

(Figure 9)

Effect of Variable temperature on Enzyme Activity

0.030
0.025
0.020
4 degrees C

% Amylose 0.015

22 degrees C
37 degrees C

0.010

70 degrees C
0.005
0.000
0

Time (min.)

References
Madden, Charles R. "Ch 6 Enzyme Activity." Cell Structure and Function: A Labratory Manual.
By Donna M. Fox. Revised Third Edition ed. Dubuque: Kendall Hunt, 2001. 63-63,70.
Print.
Sadava D.,Berenbaum, May R., H. C. Heller, and D.M Hillis. Life. The Science of Biology. Tenth
Edition ed. Sunderland, MA: Sinauer Associated, 2011. Print.
Tester, J. Karkalas and X. Qi (2004). Starch structure and digestibility Enzyme-Substrate
relationship. World's Poultry Science Journal, 60, pp 186-195. doi:10.1079/WPS200312.