BEANO©: Chemistry, Enzymes, and Flatulence
Aaron Jacobs
Lab Partner
Mustafa Hammudi
T.A.
Kate O’Rourke
Date of Experiment
1/31/2013
Due Date
2/28/2013
Introduction:
Our body is a complex network, full of processes and reactions that help keep us alive
and functioning. Most of reactions in the body require energy to take place and surpass the
energy barrier. Heat seems like a reasonable option to obtain the energy necessary, but the
problem with heat is that it not only heats up the target reaction, but all the reactions in that area.
The body, therefore, uses a catalytic resource to speed up specific reactions. The body uses
enzymes, which are catalytic proteins made by living cells, that lower the energy barriers of
biochemical reactions in the body. By lowering the activation energy needed for a reaction to
proceed, the reaction is sped up and requires less energy to commence1. Enzymes also act with a
key and lock mechanism, this means that the enzyme will only work with a specific substrate.
Enzymes use an active site, which is the ‘lock’ in this system because it only allows for a certain
substrate, or the ‘key’, to be allowed in2. Unlike heat, which provides a mean for all reactions to
commence, enzymes can target specific molecules and only allow certain reactions to take
place1. There are also rate-determining factors that delegate the speed of the enzyme reactions.
Since enzymes are catalysts, they are not used up in a reaction; this means that other
factors determine the rate of catalytic reactions. Rate of diffusion is a factor that can govern he
rate of enzyme activity7. Another factor that determines the rate of these reactions is the
concentration of the substrate. The principle behind this is that more substrate molecules will
increase the frequency of enzyme-substrate interactions. This will eventually reach a limit; the
limit is the amount of enzyme present because each enzyme’s active site is being occupied. The
temperature and the pH determine the efficiency of an enzyme1. Multiple rules of kinetics
explain the reason why temperature affects enzyme activity. By increasing temperature, there
are more energetic collisions and just more collisions in general. The collisions that are
2 occurring, increases the interaction between the enzyme and the substrate3. It must be taken into
account though, that every enzyme is different and certain enzymes work better and certain
temperatures. When enzymes work at temperatures that are not in their optimal range, the
enzyme’s activity practically cease because the enzyme will begin to denature and will stop
functioning1. Just like temperature, enzymes have optimal ranges for pH. pH can alter the amino
acid bonds, therefore altering the shape of the enzyme. The pH can also change the properties of
the substrate, which can stop binding from occuring. Pepsin for example, is located in our
stomach, which function in very acid conditions. Compare that to carbonic anhydrase, which is
active in the basic conditions of the cytosol4. Enzymes are crucial for the body to function and
problems with bodily enzymes can cause health problems.
Many processes of the body require the catalytic properties of enzymes and when a
problem arises or these enzymes aren’t present the body is negatively affected, and sometimes in
very drastic ways. Gaucher’s Disease is a genetic disorder caused by a lack of the enzyme
glucocerebrosidase. This deficiency causes lipids to be stored in blood cells, this can lead to
pain, anemia and can even lead to neurological disorders. The best way to treat this is to undergo
enzyme replacement therapy5. Another disorder caused by enzyme deficiencies is lactose
intolerance. Lactose intolerance is occurs when the body lacks proper amounts of the enzyme
lactase, which the body requires to breakdown the sugar lactose. When lactase isn’t present and
a person consumes milk, the symptoms can vary from abdominal cramps and bloating to diarrhea
and flatulence6. This experiment focused primarily on the body’s inability to breakdown certain
sugars because of our lack of a certain enzyme7.
Flatulence, which is the gas that is expelled from the rectum, is caused by multiple things
including swallowing gas, intestinal bacteria, lactose intolerance and from certain foods8. In
3 particular, indigestible sugars called oligosaccharides. Foods like peas, beans and corn contain
these sugars and our body doesn’t have the capability to digest them. The enzyme necessary to
break down these sugars is α-galactosidase and sucrose. An over the counter drug known as
BEANO© provides the body with these necessary enzymes. This lab focuses on the use of this
product in various conditions, specifically the increase or decrease in temperature as well as
substrate concentration7. As temperature is increased, it is expected that the rate of the reaction
will increase as well. It is also expected that is the concentration of the substrate, pea extract, is
increased, the rate of the reaction will also increase. And finally, as the pH is lowered, it is
expected that the rate of the reaction will increase.
Procedure:
This procedure came from the Chemistry 133B Lab Manual7. Mustafa Hammundi’s lab
manual was used as a reference9. Before beginning this lab, a general understanding of
micropipetting must be known. To practice this technique 1mL of distilled water is measured
out with the micropipette and released onto a weigh boat. Since water has a density of 1g/mL,
the balance should read 1 gram. This must be repeated five times in order to calculate the
average and standard deviation of the data, as seen in table 1 and the formulas that follow. The
next step is to calibrate the glucometer, which is done by using solutions of known glucose
concentration. This was done with five solutions, the data can be found in table 2. After the
calibration step, the experiment actually can begin. The first experiment focuses on how the
various substrate concentrations can effect the reaction rate. Begin by micropipetting 1mL of
100% pea extract into a beaker, which is then placed on a stir plate with a magnetic stir rod
inside. 1mL of BEANO is then added into the beaker, then, in two-minute increments, use the
glucometer to attain the glucose concentrations. If the glucometer reads low, keep taking
4 readings until a value appears and continue until 5 readings have been taken. These steps are
then repeated for 50% and 25% pea extract concentrations, the data for these can be seen in table
and figure 3. The second factor that is being tested is how varying temperature effects enzyme
activity. One will first fill a Styrofoam cup with hot water and adjust the temperature until it is
around 40oC. Place a 10mL beaker filled with 50% pea extract in the warm water and place the
setup on the stir plate with a magnetic stir rod inside. Add BEANO into the extract and use a
glucometer to measure the concentrations. Measure first at 1min and then in two-minute
increments starting at 4min. This process will then be repeated for the 10oC trial, but the
reaction will likely take a long time to occur so check in two-minute increments until the
glucometer doesn’t read low and record five measurements after that point. The 25oC readings
can be taken from the 50% pea extract concentrations from the previous experiment.
Results:
Table 1. Determining the Accuracy of Micropipetting.
Trial #
Mass (g)
1
0.9875
2
0.9969
3
0.9941
4
0.9914
5
0.9934
0.9875+0.9969+0.9941+0.9914+0.9934
= 0.9927
5
Average: Standard Deviation:
!.!!"#!!.!"#$ ! ! !.!!"#!!.!!"! ! ! !.!!"#!!.!!"# ! ! !.!!"#!!.!!"# ! ! !.!!"#!!.!!"# !
(!!!)
Table 2. Calibration of the Glucometer.
Trial #
Glucose Concentration (mg/dL)
1
50
2
100
3
150
4
200
5
250
=0.001467
Glucometer Reading (mg/dL)
59
153
283
372
418
5 Table 3. The Effect of Varying Substrate Concentrations on Glucose Concentrations.
Percent Pea Extract
Time (min)
Glucose Concentration (mg/dL)
4
43
6
79
8
111
100%
10
164
12
206
4
27
6
55
50%
8
82
10
110
12
144
7
22
9
35
11
45
25%
13
60
15
80
The Effect of Varying Substrate Concentrations
on Reaction Rate
Glucose Concentration (mg/dL)
250
y = 20.55x - 43.8
y = 20.55x - 43.8
R² = 0.99242
200
y = 14.45x - 32
yR²==14.45x
- 32
0.99796
150
y = 7.05x - 29.15
= 0.98362
yR²
= 7.05x
- 29.15
100
100% Pea
100% Pea
Extract
Extract
50% Pea
Extract
25% Pea
Extract
50
0
2
4
6
8
10
12
14
16
Time (min)
Figure 1. This graph shows the relationship between changing pea extract concentrations and the
reaction rate of the enzyme, it was made using data from table
6 Table 4. The Effect of Varying Temperatures on Glucose Concentrations.
Temperature
Time (min)
Glucose Concentration (mg/dL)
4
87
6
148
40°C
8
213
10
262
12
339
4
27
6
55
25°C
8
82
10
110
12
144
11
23
13
25
10°C
15
38
17
44
19
57
400
400
The
The Effect
Effect of
of Varying
Varying Temperatures
Temperatures on
on Reaction
Reaction
Rate
Rate
y=
30.9x
- 37.4
y=
30.9x
- 37.4
R² = 0.99649
Glucose Concentration (mg/dL)
Glucose Concentration (mg/dL)
350
350
300
300
40°C
250
250
40°C
25°C
200
200
y = 14.45x
y = 14.45x
- 32 - 32
R² = 0.99796
150
150
100
100
25°C
10°C
y = 4.35x - 27.85
y = 4.35x - 27.85
R² = 0.95907
10°C
5050
0 0
2 2
44
66
88
1010
1212
1414
1616
1818
2020
Time(min)
(min)
Time
Figure 2. This graph shows the relationship between changing the temperature of the solution
and the reaction rate of the enzyme; this was made using data from table 4.
7 Table 5. The Reaction Rates for Various Substrate Concentrations.
Pea Extract Concentration
Rate of Reaction (mg/dL per min)
100%
20.55
50%
14.45
25%
7.05
Table 6. The Reaction Rates for Various Temperatures.
Temperature (°C)
Rate of Reaction (mg/dL per min)
40
30.9
25
14.45
10
4.35
Discussion:
Some conclusions could be drawn from the data presented. The data for varying
substrate concentration and its effect on glucose concentrations, as shown in table 3, shows a
strong relationship of increasing glucose concentration increases the glucose concentration
readings. This relationship can be better seen in figure 1, the graph shows that the increase in
pea extract concentration increases both glucose concentrations and the rate that the glucose
concentrations increase. The reaction rates are further displayed in table 5, for 100% pea extract
concentration the rate of the reaction was at 20.55mg/dL per min, which were taken from the
slopes of the best-fit lines of figure 1. The reaction rate of 100% pea extract is almost 3 times as
high as 25%, which was 7.05, and is almost 1.5 times higher than the 50% pea extract. This
means that at higher concentrations of substrate, the α-galactosidase enzyme has an increased
reaction rate. This agrees with the hypothesis and the information stated in the introduction that
increasing substrate concentrations would increase the amount of enzyme-substrate interactions,
increasing reaction rate1.
Another conclusion drawn from the data presented is that increasing temperature also
increases reaction rates. Table 4 shows the data collected, and it clearly shows a rise in glucose
concentrations as temperature is increased. This same relationship can be better seen in figure 2,
8 which shows the graph of table 4. The rates of these reactions were drastically different, as seen
in the slopes of the best-fit lines, which also is the same as the rate of the reactions. The rates of
the reactions for the various temperatures can be found in table 6. At 40oC the rate of the
reaction was 30.9mg/dL per min, which is a huge rate increase when compared to the rates at the
other two temperatures. At 25oC the rate was 14.45, which is more than two times less the rate
of 40oC. 10oC was extremely low; a reading wasn’t picked up until 11th minute, which means
that this was a very slow reaction, which was confirmed with a rate of 4.35. There is a
significant difference in reaction rates that can be seen in at the various temperatures tested. This
data agrees with the hypothesis that as temperature increase, up to a point, the rate of an enzymes
activity will also increase. Temperature increasing causes molecules to move more rapidly,
which can increase reaction rates since the molecules will be interacting more often and at higher
speeds1. Even though pH was not tested in this lab, future experiments could support or reject
the hypothesis that a lower pH would increase the activity of α-galactosidase. The BEANO©
website wasn’t clear, but it does state that the reason indigestible foods aren’t absorbed in the
small intestine is because of a lack of enzymes. Since the website refers to the small intestine,
which is a basic environment, one would assume that α-galactosidase is active in the basic
conditions of the small intestine rather than the acidic conditions of the stomach10.
Conclusion:
The evidence presented in this lab report supported the hypotheses; both substrate
concentration and temperature have a major effect α-galactosidase’s ability to catalyze the
breakdown of oligosaccharides into glucose. The data shows that at higher substrate
concentrations, α-galactosidase has a higher reaction rate and the same trend was seen with
increasing temperature data. These results were determined through experiments that utilized
9 various pea extract concentrations and the rate that α-galactosidase’s could break down the
indigestible sugars into glucose. The same type of experiment was used to get the temperature
data, except various temperatures were substituted in for substrate concentrations.
10 References:
1. Reece, J. B., Urry, L., Cain, M., Wasserman, S., Minorsky, P., and Jackson, R. Campbell
Biology 9th Edition; Pearson Benjamin Cummings: San Francisco, CA 2011; pp 153- 156.
2. Brown, Theodore L., Lemay, H. Eugene Jr., Bursten, Bruce E., and Murphy, Catherine J.
Chemistry The Central Science 11th Edition; Pearson Education, Inc.: Upper Saddle River,
NJ 2009; pp 609
3. http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm
4. http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm
5. Somaraju UR, Tadepalli K. Hematopoietic stem cell transplantation for Gaucher disease.
Cochrane Database of Systematic Reviews 2012, Issue 7.
6. http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001321/
7. Chem 113B lab manual. Spring 2013, by William Charette Jr. published by Hayden Mcneil,
pgs (6-1)—(6-16).
8. http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/pages/Flatulence
9. Hammudi, Mustafa, Chem 113B Laboratory Notebook, pp10-14.
10. http://www.beanogas.com/en/about-beano/beano-faqs.aspx
11. Jacobs, Aaron, Chem 113B Laboratory Notebook, pp8-12.
11