Fall

Chris Messner
Chemistry 113B Beano Formal Lab Report
February 21st, 2015

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Introduction:
The human body functions thanks to numerous chemical
reactions. The forces behind such chemical reactions, propelling them
forwards and increasing their speed and efficiency, are enzymes.
Enzymes are, protein-based molecules that catalyze biochemical
reactions (1). The reason enzymes are so useful is that they lower the
activation energy required for a reaction to take place, allowing more
of the reaction to occur for less energy expenditure (2). Every reaction
requires an input of energy (the aforementioned activation energy) to
begin, so enzymes are what allow many chemical processes to occur.
Enzymes work using a termed, lock-and-key method (3). The
idea behind lock-and-key is that an enzyme has a specific molecule
that fits into it, a substrate. Only when that substrate molecule in its
specific shape and size enters the enzymes active site, does the
enzyme perform its function. An active site is the specific region of
the enzyme which combines with the substrate (3). The enzyme is
the metaphoric lock, which will only open (function) when the proper
key (the substrate) enters the keyhole (the active site). However it is
believed that the substrate may possibly cause change in the shape of
the enzyme. The induced-fit theory says that enzymes are flexible,
and adjust their active site shape to fit their substrates (3).
The experiment performed was a series of tests, with hopes to
better understand properties of enzymes. The tests involved the

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manipulation of a certain variable, while keeping the others constant,
and then observing the reaction rate of enzyme and substrate, relative
to the changed variable. The two variables tested were substrate
concentration and temperature. Using a glucometer (more specifically
a ReliOn Ultima) (4), it was determined what a change in each of these
variables would do to the amount of glucose produced in a set time
frame from the enzymatic reaction of -galactosidase and
oligosaccharides. Specifically, the oligosaccharides found in peas.
Oligosaccharides are carbohydrates from 2 to 20 monomeric units
long that are not naturally broken down in the body, but are instead
fermented, producing gas (5). The enzyme, -galactosidase, breaks
down the pea oligosaccharides to produce -glucose, which
interconverts into -glucose. -glucose is put onto the glucometer test
strip, and the enzyme converts it into gluconolactone, which is
detected by the glucometer (4).

Procedure (4):
The procedure involved the usage of Beano, a product taken to
help with excessive gas problems, because it contains the
aforementioned -galactosidase enzyme, which breaks down the
oligosaccharides that would otherwise cause flatulence (release of
excess the gases built up during fermentation). However before any
testing could be done the glucometers were calibrated to work in water

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matrices, instead of blood. To do so predetermined quantities of
dextrose were put into equal volumes of water, which were then tested
by the glucometer. The addition of dextrose to water was quantified by
dividing the mass (in milligrams) by the volume (in deciliters).
After the calibration was complete, testing for the effect of
varying levels of substrate began. 100%, 50%, and 25% pea extract
were taken and placed into a 10-milliliter beaker. The beaker was
placed onto a stir plate, and using a magnetic bar the solution was
stirred at a slow speed. 1.000 milliliters of Beano was taken by
micropipette and put into the stirring solution. Every two minutes each
solutions glucose level was tested via glucometer in units of
milligrams per deciliter, until five usable data points were found.
The second variable tested was temperature. 1 milliliter of 50%
pea extract was put into a 10-milliliter beaker. The beaker was moved
into a Styrofoam cup that was 3-centimeters full with 40 degree Celsius
water, and then covered to prevent heat loss. 1-milliliter of Beano was
added, and each minute the glucose level was measured. This was
repeated with a cup that contained 10 degree Celsius water. The
results were compared to the original 50% pea extract reaction, which
was at about 25 degrees Celsius, room temperature.

Results (6):
Glucose Calibration Test

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Concentration Added (mg/dL)
50
100
150
200
250

Glucometer Reading (mg/dL)

65
130
221
259
327

Variable 1: Substrate Concentration

100% Pea Extract
Time (minutes)
4
6
8
10
12

Glucometer Reading (mg/dL)

76
97
147
174
230
50% Pea Extract

Time (minutes)
10
12
14
16
18

Glucometer Reading (mg/dL)

45
66
68
86
74
25% Pea Extract

Time (minutes)
10
12
14
16
18

Glucometer Reading (mg/dL)

29
25
40
40
36

Variable 2: Temperature
40 Degrees Celsius

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Time (minutes)
4
6
7
8
9

Glucometer (mg/dL)
25
60
64
85
95
10 Degrees Celsius

Time (minutes)
26
28
30
32
34

Glucometer (mg/dL)
20
29
38
33
47

Discussion:
The glucometer calibration results themselves were high,
however the trend of glucose increase was expected and ran parallel
with the actual glucose values. The information gave insight as to how
accurate the glucometer truly was. While inaccurate, the values
followed the trend appropriately.
Before data values for the substrate and temperature
experiments can be compared, there must be some data removal to
compensate for error. The data to be removed are not exorbitant
outliers, however the data does not make sense within the realm of the
experiment. The data are very clear errors. It is very possible these

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errors were due in part to faulty measurement procedures. This could
include poor sterilization of pipet tips, the tools used to transfer the
solution to the glucometer, time miscalculations, and other
misinterpretations. One other possibility is a faulty glucometer or
faulty glucometer test strips.
The data to be removed are as follows. In the 25 degrees Celsius
50% pea extract data table, the 18-minute value should be disregarded
because the glucose concentration somehow decreased, which in the
context of this experiment is highly unlikely. The 12-minute and 18minute concentration values should be removed for the 25% pea
extract as well. This is because the 25 mg/dL found at 12 minutes was
a decrease in concentration from the previous 29, and the 36 mg/dL
found at 18 minutes was a decrease in concentration from the previous
40. Finally, in the 10 degrees Celsius data chart, the 32-minute
concentration went down from the previous concentration of 38.
The results of the substrate concentration manipulation correlate
with the idea that more substrate allows for faster reaction rates and
more products. Comparatively the 100% pea extract produced much
more glucose than the 50% or the 25%, and the 25% produced the
least amount of glucose. At 12 minutes, the 100% had produced 230
mg/dL, the 50% produced 66 mg/dL and the 25% produced only 25
mg/dL. More importantly however, the slopes of the three sets of data
correlate as well. The slope gives the concentration production over

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time, and through slope it is clear to see the rates at which each
solution is producing glucose. To obtain the slope each consecutive
data value must be added to the previous, and then divided by the
time, giving a unit of milligrams per deciliter per minute. For example
at the 4-minute mark, the 100% pea extract had 76 mg/dL. The
solution began with zero, so (76-0) mg/dL divided by 4 minutes, will
give 19 mg/dL/min. This step is repeated for all valid data points,
which in this case is five. The values are then added, and the total is
divided by five giving slope of 19.2 mg/dL/min for the 100% pea
extract concentration values. For the 50% pea extract the slope is
6.25 mg/dL/min, and for the 25% pea extract the slope is 1.9
mg/dL/min. The 100% produces the most glucose, the fastest, and the
25% produces the least. This is as expected, and portrays solutions
that contain higher concentrations of substrates react faster.
The reason that higher concentrations of substrate make
products with more speed has to do with the fact that when there is
more substrate present, more reactions can occur at one time. E + S
= ES = E + P is the equation that represents this, where an enzyme
plus a substrate becomes an enzyme-substrate complex, which then
becomes and enzyme and a product. The more substrate, the more
enzymes can be filled, which then move on to the next substrate.
Initially the rate rises rapidly with increasing substrate concentration.
Above a certain concentration, however, all active sites are occupied,

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and the reaction becomes zeroth order in the substrate (7). An
increase in the amount of substrate will increase product yield speed,
up until a certain point where there are no more enzymes. Then and
only then will additional substrates have no effect on production speed,
because there are no more enzymes available for them to react with.
Reaction speed will stay consistent, as the substrates wait for the next
enzyme to open up its active site.
Now, based on the temperature manipulation results it is clear
increasing temperature increases the reaction speed of enzymes.
Comparison of slope reveals this in a manner similar to that of the
substrate data. The slope for the 40 degrees data points is calculated
just as it was for the substrate data, and it is 11.6 mg/dL/min. This is
higher than the 25 degrees slope, which was previously calculated as
6.25 mg/dL/min. The 10 degrees slope is also lower than the 25, with a
slope of 3 mg/dL/min.
Reaction rate increases at higher temperatures because of
energys effect on molecules. The function of an enzyme is to lower
the required activation energy for a substrate to go through a reaction.
When temperature is added, more energy is added. The rate of a
reaction will increase if the number of molecules with enough energy
to provide the activation energy of the reaction increases (8).
Enzymes and increased temperature work together to make it very
easy for chemical reactions to occur. However, with the addition of

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heat (similarly to the addition of substrate) enzymatic activity will only
increase to a point. At higher temperatures the enzyme will denature
and the bonds making it up will break apart. Most enzymes denature
between 40 degrees and 70 degrees Celsius, which this experiment
was approaching (1).

Conclusion:
Based on experimental data increasing substrate concentration
and increasing temperature both increase the speed of enzymatic
reactions. Beano and the oligosaccharides found in pea extract
accurately represented biochemical interactions between substrate
and enzyme. The -galactosidase enzyme from Beano functioned
appropriately under the scope of the experiment, and is shown to be
effective in managing the break down of indigestible sugars. When
more oligosaccharides were present, there was a faster reaction rate
as more sugars were available for reaction. When there was a
temperature increase, the energy of the solution increased, and so a
greater number of oligosaccharide molecules possessed the necessary
activation energy to react.

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References:

1. Cain, M. L.; Bowman, W. D.; Hacker, S. D. Ecology Third Edition;

Sinauer
Associates, Inc.: Sunderland, 2014; p 90.
2. Farabee, M. J. Reactions and Enzymes. Online Biology Book.
http://www2.estrellamountain.edu/faculty/farabee/BIOBK/BioBook
Enzym.html (accessed Feb 22, 2015).
3. Ophardt, C. E. Mechanism of Enzyme Action. Virtual Chembook.
http://www.elmhurst.edu/~chm/vchembook/571lockkey.html
(accessed Feb 22, 2015).
4. Messner, C., Chem 14 Laboratory Notebook, pp 6-10.
5. Slavin, J.; Roberfroid, M. Nondigestible oligosaccharides. PubMed.
http://www.ncbi.nlm.nih.gov/pubmed/11186236 (accessed
Feb 22,
2015).
6. Charette Jr. William, Chemistry 113B Laboratory Manual, Spring
2015,
Hayden-McNeil, 2015, pp 6-1 - 6-16.
7. Burdge, J.; Overby, J. Chemistry Atoms First; McGraw-Hill: New
York, 2012; pp
591 to 592.
8. Rogers, E.; Stovall, I.; Jones, L.; Chabay, R.; Kean, E.; Smith, S.
The Rate of

Reaction. Fundamentals of Chemistry.

http://chem.wisc.edu/deptfiles/genchem/sstutorial/Text13/Tx133/t
x133.html (accessed Feb 22, 2015).