Various Factors on the Enzymatic Activity of Beano

Matthew Mouck
Partner: Hannah Odenwald
CHEM 113B Section: 003
1 March 2018
Aneesa Bokhari
Introduction:

In living systems, chemical reactions constantly occur and allow life to continue.

Chemical reactions occur when there are collisions between molecules and when these molecules

have substantial energy to overcome the activation energy1. With this, the orientation of the

molecules must be in a certain manner in order for the reaction to place. Furthermore, it is

important to note that the energy needed for the reaction to occur by reaching the activation

energy is referring to the kinetic energy of the molecules. Chemical reactions are constantly

occurring, but it is necessary that they occur at a certain rate in order for life to be sustained.

Kinetics studies the rates of reactions. It is known there are several methods for

increasing the rates of reactions. For instance, increasing the temperature of the reactions

increases the overall kinetic energy of the molecules and thus allows more collisions to occur

that have the necessary activation energy. In effect, increasing the temperature will cause the rate

of the reaction to increase and decreasing the temperature will cause the rate of reaction to

decrease. Along with the temperature, increasing the concentration of the reactants will increase

the frequency of collisions. Yet again, more collisions will cause the rate of the reactions to

increase. Thus, increasing the concentration of reactants will cause the rate of reaction to

increase while decreasing the concentration will decrease the rate2.

Perhaps most important, the introduction of a catalyst will also increase the rate of the

reaction. A catalyst will typically take the form of an enzyme, which is a protein. It does not

change the energy difference between the reactants and products. An enzyme is of particular

interest to biological study and kinetics because an enzyme functions by reducing the activation

energy needed for a reaction to occur but it is not consumed in the process. This means that an

enzyme can catalyze multiple reactions and thus rapidly increase the rate of a reaction3.

1
 However, it is important to note that enzymes are strongly influenced by the environment

around them. First, an enzyme is proposed to function like a lock-and-key with the substrate that

binds to it. However, it is also considered to function as induced fit. Regardless, the core theme

is that an enzyme will slightly alter its shape so that the substrate is able to bind to the enzyme.

This is significant as it demonstrates that enzymes are selective with what substrate is able to

bind to it. A certain enzyme will only allow certain substrates to bind, thus a given enzyme

cannot catalyze every reaction that occurs in the body but only specific reactions. Furthermore,

an enzyme is said to have an optimal range of function in relation to both temperature and pH.

There is a peak temperature and peak pH for every enzyme. However, when the temperature and

pH stray from the peak, the efficiency of an enzyme begins to diminish and the rate of reactions

will decrease. Furthermore, if the temperature and pH move beyond the optimal range, the

enzyme will denature. A denatured enzyme is permanently incapable of catalyzing future

reactions. One further effect of enzyme efficiency is oversaturation of substrate. If the

concentration of substrate becomes increasingly large, the enzyme will reach a peak in its

efficiency and adding more substrate will not cause the reaction rate to increase any further4.

Enzyme deficiency in the body is an issue for some individuals. Because enzymes reduce

the activation energy and are essential for many chemical reactions to occur, lacking an enzyme

likely draws adverse consequences. For instance, lacking the enzyme beta-galactosidase cannot

break down lactose into galactose and glucose. This causes the condition known as lactose

intolerance that prevents consumption of foods with lactose unless a specific method to acquire

beta-galactosidase through artificial means is undertaken, such as buying medicine or specialized

milk5.

2
 Beano is an example of a medicine that can be used when enzymes are missing in the

body. Beano is a product that contains an enzyme that can break down oligosaccharides. Foods

with oligosaccharides are beans, asparagus, cauliflower, cabbage, among others. The body

cannot break down oligosaccharides for some people, so foods containing this are not digested

properly. This results in flatulence as the bacteria break down the foods and release a gas. The

buildup of the gas causes the flatulence. Beano is able to reduce the expression of flatulence and

limit the gas buildup since it contains an enzyme that allows a certain chemical reaction to occur.

Beano contains the enzyme alpha-galactosidase, which is capable of breaking down the

oligosaccharides into galactose and sucrose. Sucrose can be broken down by the enzyme sucrase

into fructose and alpha-glucose. Therefore, it is observable that when an oligosaccharide is

completely broken down, one of its final products is glucose. Glucose can be measured by test

strips6.

Beano has been studied and it appears that it is effective in reducing the flatus produced

as compared to a placebo pill taken7. It does appear that the data is statistically significant.

Therefore, it is imperative to understand the chemistry of the product since it is clearly having an

effect on the body. This experiment focuses primarily on the effects of various external factors

on the enzymatic activity of Beano.

This experiment aimed to investigate the aforementioned factors that affect the enzymatic

activity of alpha-galactosidase in Beano. Specifically, the factors of substrate concentration and

temperature were examined. The substrate concentration was evaluated by varying the percent of

pea extract that was utilized and a second experiment manipulated temperature, as the

temperature was changed 15°C higher and lower from the starting 25°C. Based on the outside

information, the following hypothesis consisting of two parts was created: if the percent of the

3
pea extract is decreased, the rate of the reaction will decrease; furthermore, if the temperature is

raised the rate of the reaction will increase whereas if the temperature is decreased, the rate of the

reaction will decrease.

Procedure:

The procedure for this experiment comes from the PSU Chemtrek7.

A ReliOn Ultima glucometer was obtained to measure the glucose. It was calibrated by

using known concentrations of glucose solutions and reading the output of the glucometer.

A Beano solution comprised of 1 tablet dissolved in 50 mL of water and 10 mg invertase

was used. 1.000 mL of 100% pea extract was added to a 10 mL beaker and a stir bar was placed

inside the beaker. The beaker was placed onto a stir plate, where the setting was turned onto low

speed. Using a thin-stem pipet, 1.000 mL of Beano was added into the beaker. After two

minutes, the pipet stem was dipped into the beaker and touched to the stem of the glucometer

strip. The reading was recorded and the stem of the pipet was cut off to prevent contamination of

future readings. Four more readings were taken (for a total of five readings), each with a new test

strip for the glucometer. The readings were taken for two-minute intervals. Using clean beakers

and stir bars, this process was repeated by obtaining 1.000 mL of 50% pea extract and 25% pea

extract. However, the first glucose reading for the 50% pea extract and 25% pea extract did not

occur until six minutes into the experiment. Thereafter, the final four recordings for each pea

extract concentration were taken for every subsequent two-minute intervals. Once all the data

was obtained, graphical analysis can be completed by creating a glucose concentration versus

time for each pea extract.

4
 The next part of the procedure focused on different temperatures. One regular and one cut

Styrofoam cup were obtained. The regular Styrofoam cup was filled with hot water and ice was

added until it reached 40°C. A 10 mL beaker was placed into the cut Styrofoam cup and a

magnetic bar placed into the beaker, with the whole system being centered on the stir plate. The

40°C water was placed around the beaker. 1.000 mL of the 50% pea extract was poured into the

10 mL beaker. The stir plate was turned onto quick rotation, but not so quick so that the solution

splashes. 1.000 mL of Beano was added and the setup was covered by the regular Styrofoam cup

for heat retention purposes. After one minute, a reading with the glucometer was taken. Reading

were taken with new test strips each subsequent minute until five total readings were obtained.

All readings were recorded. Using a new beaker and stir bar, the exact situation was replicated

but with 10°C water instead of 40°C. However, the first reading did not occur until six minutes

of the Beano being added and subsequent readings occurred every two minutes until a total of

five readings was obtained. All readings were recorded. A graph of glucose concentration versus

time was created with the various temperatures.

5
Results:

The following results are from my chemistry notebook8, Matthew Mouck, from pages 8-11 and

Hannah Odenwald’s chemistry notebook9 (pages unknown).

Table 1. Glucose Concentration for Varied Pea Extract Concentrations

Recorded Glucose Concentrations (mg/dL)

Times (minutes) 100% Pea Extract 50% Pea Extract 25% Pea Extract

2 55 - -

4 105 - -

6 243 69 43

8 414 117 59

10 577 146 75

12 - 190 94

14 - 217 111

6
Graph 1. Glucose Concentration Versus Time for Various Pea Extracts. The slopes of the graph

depict the reaction rate for each pea extract concentration. Data for points came from Table 1 and

the graph was created and the slope was calculated through Excel

Glucose Concentrtion vs. Time For Various Pea Extracts

900

800 y = 67.65x - 127.1

Glucose Concetnration (mg/dL)

700

600

500

400

300

200 y = 18.45x - 36.7

100 y = 8.55x - 9.1

0
0 2 4 6 8 10 12 14 16
Time (minutes)

100% Pea Extract 50% Pea Extract 25% Pea Extract

Linear (100% Pea Extract) Linear (50% Pea Extract) Linear (25% Pea Extract)

7
Table 2. Glucose Concentration for Various Temperatures

Recorded Glucose Concentrations for 50% Pea Extract (md/dL)

Time (minutes) 40°C 25°C 10°C

1 44 - -

2 78 - -

3 111 - -

4 162 - -

5 218 - -

6 - 69 40

8 - 117 50

10 - 146 63

12 - 190 79

14 - 217 97

8
Graph 2. Glucose Concentration Versus Time for Various Temperatures. The slopes of the graph

depict the reaction rate for the various temperatures. Data for points came from Table 2 and the

graph was created and the slope was calculated through Excel

Glucose Concentration vs Time for Various Temperatures

700

600 y = 43.2x - 7
Glucose Concentration (mg/dL)

500

400

300

y = 18.45x - 36.7
200

100
y = 7.15x - 5.7
0
0 2 4 6 8 10 12 14 16
-100
Time (minutes)

40°C 10°C 25°C Linear (40°C) Linear (10°C) Linear (25°C)

Table 3. Reaction Rates for Various Pea Extracts

Pea Extract Concentration Reaction Rate (mg/dL per minute)

100% 67.65

50% 18.45

25% 8.55

9
Table 4. Reaction Rates for Various Temperatures

Temperature (°C) Reaction Rate (mg/dL per minute)

40 43.2

25 18.45

10 7.15

Pea Extract Reaction Rate Ratios:

𝑚𝑔
67.65
𝑑𝐿∗𝑚𝑖𝑛
100% to 50% = 𝑚𝑔 = 3.67
18.45
𝑑𝐿∗𝑚𝑖𝑛

100% to 25% = 7.91

Discussion:

The obtained results support the hypothesis that decreasing the substrate concentration

decreases the rate of the reaction and increasing the temperature increases the rate of the

reaction, whereas decreasing the temperature decreases the rate of the reaction.

Decreasing the substrate (pea extract in this case) does appear to decrease the rate of the

reaction (and vice versa—increasing the concentration increases the rate). This is evidenced by

examining the ratio of the rates for various concentrations. The ratio of the rate of the 100% pea

extract to the 50% pea extract is 3.67. This means that the rate of the reaction of the 100% pea

extract occurred 3.67 times faster than the 50% extract. And by looking at the 100% pea extract

to the 25% pea extract ratio of rates, 7.91 emerges. This means that the rate of the 100% pea

extract occurs 7.91 times faster than the rate of the 25% pea extract. Furthermore, a simple

quantitate comparison can be done by looking at the slopes alone. The magnitude of the slope of

the 100% pea extract is 67.65, of the 50% is 18.45, and of the 25% is 8.55. It is obvious to see

10
that the rate of the reaction decreases as the concentration of pea extract (substrate) decreases.

The likely reason that the higher reaction rates are seen are because there is more substrate

available to bind to the enzyme. The greater the concentration the substrate, the more reactions

can occur until the enzyme reaches its peak efficiency. The enzyme is able to break down the

oligosaccharides found in the pea extract at a greater rate for the higher concentration of pea

extract since more substrate binds faster to the enzyme. This increases the rate of breakdown of

the oligosaccharides, thereby increasing the concentration of glucose at a faster rate which gives

the overall higher readings from the glucometer for the higher pea extract.

Decreasing the temperature appears to decrease the rate of the reaction. The 25°C had a

rate value of 18.45, whereas the 10°C had a rate value of 7.15 (both are 50% pea extract

concentration). Clearly, since only the temperature is different, the difference in slope can be

attributed to the change in temperature. Thus, it seems a lower temperature decrease the rate of

the reaction. Because the slope is a measure of the enzymes activity, the lower temperature can

thus be extended to causing a decrease in enzymatic activity. The reason the enzymatic activity

(and thus the rate of reaction) is lower is because the lower temperature gives a lower overall

kinetic energy; thus, there are fewer collisions with the enzyme. It is also possible the enzyme

becomes less flexible at lower temperatures, thus causing less opportunity for the substrate to

bind to the enzyme10. This causes the rate of oligosaccharides broken down to decrease, thus

lowering the rate of glucose emergence. This is likely what accounts for the lower glucose

readings at lower temperatures.

Increasing the temperature causes the rate of reaction to increase. The 40°C had a rate

value of 43.2, whereas the 25°C had a rate value of 18.45 (both are 50% pea extract

concentrations). Clearly, since only the temperature is different, the difference in slope is due to

11
the change in temperature. Thus, it seems a higher temperature increases the rate of the reaction.

Because the slope is a measure of the enzyme activity, the higher temperature can thus be

extended to causing an increase in enzymatic activity. The reason the enzymatic activity (and

thus the rate of the reactions) is higher is because higher temperature gives a higher overall

kinetic energy; thus, there are more collisions with the enzyme allowing for more reactions. This

idea of a higher temperature increasing kinetic energy and thus reaction rate has already been

researched2. This means that the rate that the oligosaccharides are broken down will increase,

thus giving a higher glucose output since glucose is the final product of the breakdown. In fact,

an increase in temperature of 10°C can cause a 50% to 100% increase in enzyme activity11. This

particular experiment did not raise the temperature by 10°C, so it would be difficult to compare

or even estimate. However, it is consistent with the idea that increase in temperature has a drastic

effect on enzymatic activity.

It should be noted that the data collected cannot demonstrate the optimal temperature

range of the enzyme. The temperature should be increased further until the calculated rate of

reaction decreases. Then, the optimal temperature can be pinpointed by changing the temperature

less and less between trials. It would be useful to know this temperature since the digestive track

has its own temperature. Therefore, the efficiency of Beano in the body as it currently functions

can be calculated.

Although the data gathered support the hypothesis, there are parts of the experiment that

introduced error and thus skewed the results. For instance, initial struggle with the use of the

glucometer was an issue for the readings of the 100% pea extract. It is expected that the rates be

proportional to the ratio of the concentrations of the pea extract because the pea extract serves as

the substrate. Therefore, halving the substrate concentration should have the rate of the reaction.

12
However, the 100% pea extract had a rate of 67.65 mg/dL and the 50% had a rate of 18.45

mg/dL. The ratio of the two rates is about 4:1, which is double what is expected. Therefore, the

idea that there was struggling with the 100% pea extract readings is supported.

Moreover, the ice bath most certainly was not maintained at 10°C since the ice would

melt and thus allow the temperature to raise. Because the temperature is raising, the average

kinetic energy of the molecules will increase and thus increase the number of reactions with the

enzyme. Therefore, the data for the 10°C should be taken lightly because the true reaction rate

for 50% pea extract with the Beano enzyme is likely lower than what is reported. A method to

maintain the 10°C should be implemented. One possible idea is to continually add ice so the

temperature remains low, but this introduces the possibility of causing the temperature to go

below 10°C and it will still allow the temperature to rise above 10°C at some points.

In the future, experiments that examine the effects of pH should be conducted. According

to previous research, there is also an optimal range for pH for enzymes and the optimal range can

vary greatly between enzymes12. Beano works in the digestive tract13. The digestive tract can

have a pH that ranges from acidic with a pH around 4 to fairly neutral with a pH around 7.414. To

understand if the enzyme is actually as efficient as it could be in the human body, it is worth

increasing and subsequently decreasing the pH to discover the point of denaturation of the

enzyme and value of pH that gives the highest reaction rate.

Conclusion:

In general, the experiment supported the hypothesis that decreasing substrate

concentration decreases the rate of the reaction and that increasing temperature increasing the

rate of the reaction, whereas decreasing the temperature decreases the rate of the reaction.

13
According to the data collected, the reaction rate of the enzyme was higher for the higher

concentrated pea extracts and the reaction rates decreased as the temperature decreased. The core

reason is likely because the rate at which is the substrate can bind to the enzyme is altered. When

there is less pea extract, less substrate binds. When the temperature decreases, the number of

collisions likely decreases and reduces the number of reactions that can be catalyzed. Beano is an

enzyme that can aid in digestive issues for those who lack the enzyme alpha-galactosidase. The

enzyme has important functions for those who suffer from flatulence and gas buildup from eating

foods which contain oligosaccharides. Future studies investigating pH should be conducted to

further the knowledge of Beano’s enzymatic activity. Beano’s enzymatic activity is consistent

with the hypothesis, which was created using established scientific data and therefore Beano as

an enzyme follows the expected trends of an enzyme when the environment is altered.

14
References:

1. The University of North Carolina at Chapel Hill. Collision Theory: What It Takes for

Chemicals to React. Collision Theory: What It Takes for Chemicals to React.

http://cssac.unc.edu/programs/learningcenter/Resources/Study/Guides/Chemistry%20102/Collisi

on%20theory (Accessed 21 February, 2018).

2. Reaction Rates. https://www.chem.fsu.edu/chemlab/chm1046course/rates.html

Chemistry 302. http://ch302.cm.utexas.edu/kinetics/catalysts/catalysts-all.php (Accessed 20

February 2018)

3. Enzymes. RSC. RSC. http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm

http://lindblomeagles.org/ourpages/auto/2014/3/10/46966545/enzymes%20and%20beano.pdf

(Accessed 24 February, 2018)

4. Beals, M., Gross, L., and Harrell, S. (1999) Enzymes and the Rate of Chemical Reactions.

http://www.tiem.utk.edu/~gross/bioed/webmodules/enzymes.htm (Accessed 25 February, 2018)

5. (2014, June 1) Lactose Intolerance. National Institute of Diabetes and Digestive and Kidney

Diseases. U.S. Department of Health and Human Services.https://www.niddk.nih.gov/health-

information/digestive-diseases/lactose-intolerance (Accessed 24 February, 2018)

6. Ganiats, T.G., Norcross, W.A, Halverson, A.L., Burford, P.A., Palinkas, L.A. “Does Beano

prevent gas? A double-blind crossover study of oral alpha-galactosidase to treat dietary

oligosaccharide intolerance.” J-Fam-Pract. 1994 November; 39(5):441-445

7. Chemistry 113B Manual. William A. Charette, Hayden-McNeill Spring 2018: 6-1 to 6-15.

8. Mouck, Matthew. Chem 113 Laboratory Notebook: 8-11.

9. Odenwald, Hanna. Chem 113 Laboratory Notebook.

15
10. Geir Villy Isaksen, Johan Aqvist, Bjorn Olav Brandsdal. “Enzyme surface rigidity tunes the

temperature dependence of catalytic rates.” PNAS 2016 July; 113 (28) 7822-7827.

11. Introduction to Enzymes. Temperature Effects (Introduction to Enzymes).

http://www.worthington-biochem.com/introbiochem/tempeffects.html

12. The Effect of pH on Enzyme Activity.

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm (Accessed 25 February,

2018).

13. Beano FAQs. Beano Gas. http://www.beanogas.com/faqs/ (Accessed 26 February, 2018).

14. Fallingborg J. “Intraluminal pH of the human gastrointestinal tract.” Dan Med Bull. 1999

Jun; 446(3): 183-186.

16