THE SECRETS BEANO REVEALS ABOUT ENYMATIC ACTIVITY

Alaina Weinheimer

February 20th, 2013

CHEM 113B, Section 104

Partner: Rachel Thomas

TA: Kate O’Rourke

Introduction:

In the human body, thousands of chemical reactions occur every single moment.

According to the Collision Theory, which is based on the Kinetic Molecular Theory, reactions

occur when two molecules of reactant make contact. In order of these reactant molecules to

collide, each must have the minimum kinetic energy, or activation energy, and the proper

orientation when they make contact1. Some reactions in the human body can take an extremely

long time in order for the reactant molecules to overcome the activation energy and make proper

contact. To increase the rate of reactions, body cells contain enzymes that catalyze, or promote,

these reactions. If enzymes did not exist, bodily reactions would occur at such a low rate that life

would not be able to sustain2. Enzymes are proteins that contain an active site for a specific

substrate, or reactants, of the reaction. The enzyme lowers the activation energy and the reaction

occurs. The product leaves the enzyme and the enzyme finds new substrate molecules. With the

reactants together in close proximity at the proper orientation in the enzyme, decreasing the

activation energy, enzymes increase the rate of reaction3.

Many factors contribute to the rate of enzymatic activity, which effects the rate of

reaction. Some of these factors include temperature, pH, substrate concentration, and water

activity3. The effect of increasing temperature on the rate of reaction is based on the Kinetic

Molecular Theory. The Kinetic Molecular Theory states that the kinetic energy of a molecule is

directly related to temperature. If temperature increases, the speed of the molecule increases.

When the molecules of reactant have a higher kinetic energy, moving at a faster speed, contact

with an enzyme is more frequent causing the reaction to occur faster. Conversely, if the

temperature is decreased, the reactants move slower, contact is less frequent with the enzyme,

causing the reaction to occur much more slowly4. In short, enzymes are more active at higher

temperatures and less active at lower temperatures.

Varying substrate concentration relates to enzymatic activity. If there is more substrate,

or reactant, more reactant is present to bind to the enzyme and react. Thus, enzyme activity

increases when substrate concentration increases, which consequently increases the rate of

reaction. Varying enzyme concentration also affects the rate of reaction. With a lower enzyme

concentration, only so much substrate can bind to the fewer enzymes, causing the reaction rate to

decrease. With a higher enzyme concentration, more substrate can bind to the abundant

enzymes, causing the reaction rate to increase5. In summary, varying substrate and enzyme

concentration impacts enzyme activity, which directly impacts the rate of reaction.

In this experiment the enzymatic activity of the enzyme in Beano was analyzed. Beano

contains the enzyme alpha-galactosidase, which speeds up the breakdown of sugars called

oligosaccharides, which are present in foods such as peas, onions, and legumes. The enzyme,

alpha-galactosidase breaks down these oligosaccharides into galactose and sucrose. The sucrose

is then broken down by sucrase into alpha-glucose and fructose6. Many people lack the enzyme

alpha-galctosidase in their small intestine. This enzyme is located largely in the large intestine,

where bacteria, that have alpha-galactosidase, ferment the oligosaccharides and release gas as a

waste product. This gas can create a pressure on the lower stomach that may be uncomfortable

to some. To decrease the pressure by decreasing the amount of gas, people consume Beano,

which contains the alpha-galactosidase. By consuming Beano, the enzyme enters the small

intestine and can break down the oligosaccharides before they reach the large intestine7.

In this experiment, the enzymatic activity of Beano was measured based on the varying

factors. The activity was measured by a glucometer, which can only detect beta-glucose. Beta-

glucose has a different structure than alpha-glucose, which is the end product of the reaction the

enzyme induces. The location of the alpha and beta hydroxyl groups is in different places.

Luckily, in solution, alpha-glucose readily converts to beta-glucose, allowing the enzymatic

activity of Beano to be measured by the glucometer6. The glucometer used in this experiment,

the ReliOn Ultima (Wal-Mart Stores, Inc.), is designed to measure the levels of glucose in blood.

Blood contains many complex molecules. The glucometer is designed to factor in those other

molecules in its reading. In this experiment, the substrate of the enzymes in Beano was a pea

extract. Because this experiment involved a water-based solution of pea extract and Beano, not

blood, the glucometer had to be calibrated to the water so that there was a better indication of

how much glucose is in the solution.

Beano’s enzyme activity was measured for varying substrate concentration and varying

temperature in this experiment. To test for the effect of varying substrate concentration on

enzyme activity, three different solutions of pea extract were tested for glucose levels over

intervals of two minutes: 25%, 50%, and 100% pea extract. It is expected that the 100% pea

extract will lead to higher enzymatic activity, and faster rate of reaction than the lower

concentrations of pea extract, because in the 100% pea extract there are more substrate

molecules to interact with the enzymes. To test the effect of temperature on enzymatic activity,

the glucose concentration of solutions containing 50% pea extract were measured at 10ᵒC, 25ᵒC,

and 40ᵒC over two minute intervals. It is expected that the reaction carried out at 40ᵒC will yield

the fastest rate of reaction than the other two temperatures because at high temperatures,

molecules move faster. Other factors that weren’t tested for, but were noted was varying enzyme

concentration and varying pH. It is expected that with increasing enzyme concentration, enzyme

activity increases, which increases the rate of reaction. Considering that the enzyme alpha-

galactosidase is located in the human instestine, which is an acidic environment, it is expected

that there is more enzyme activity at low pH than at high pH6. In short, this experiment

measured the enzyme activity of Beano with many factors.

Procedure:

This experiment was carried out using the steps in the PSU Chemtrek6. To start, the

glucometer was calibrated to aqueous solutions containing varying concentrations of glucose.

Next, the test for the effect of varying substrate concentration on the enzyme activity

began. A solution was prepared by adding 1.000mL of 100% pea extract and a stir rod to a

20mL vial that was placed on a stir plate to mix the solution. 1.000mL of Beano solution was

added to the vial. After two minutes of mixing, the solution in the vial was measured for glucose

concentration by the placing a little sample of solution on the glucometer. After another two

minutes passed, the glucose concentration was measured again. The glucose concentration was

measured in two minute intervals until the glucometer had five numeric measurements, which

happened after 10 minutes passed in this case. This process was repeated for a solution of 50%

pea extract with Beano and 25% pea extract and Beano. Once all of the measurements were

recorded, they were graphed and a line of best fit was found for each concentration of pea extract

over time, which reflected the rate of reaction, the enzyme activity.

To test the effect of temperature on enzyme activity, special environments had to be

prepared for the vials. To achieve 40ᵒC, a Styrofoam cup was cut to fit the vial. The vial with

1.000mL of 50% pea extract and a stir rod was put into the Styrofoam cup. A separate cup was

filled with hot tap water and adjusted to be 40ᵒC. That water was then carefully poured into the

Styrofoam cup that contained the vial, without any water entering the vial. 1.000mL of Beano

was added to the vial. After two minutes intervals of mixing, the glucose concentrations of the

vial solution were measured until five numeric measurements were read.

To achieve a 10ᵒC environment for the solution of Beano and pea extract, the water bath

in the Styrofoam cup of 40ᵒC was replaced by tap water cooled by ice to be 10ᵒC. A new solution

of 50% pea extract and a stir rod were added to a clean 20mL vial. 1.000mL of Beano was

added to the vial and the glucose concentrations were measured again in increments of 2 minutes

until the glucometer read five numeric measurements. The glucose concentrations at the different

temperatures of 40ᵒC , 25ᵒC and 10ᵒC were plotted against time. The glucose concentrations

measured at 25ᵒC were used from the readings obtained during the testing of varying substrate

concentration.

Results:

Glucometer Calibration Data

Table 1. Calibration of Glucose Readings in the ReliOn Ultima Glucometer

Caption: This table shows the glucometer readings of increasing concentrations of glucose in

milligrams per deciliter. These values were then used to create a calibration curve, Graph 1.

Glucose Concentration (mg/dL) Reading (mg/dL)

50 62

100 223

150 281

200 323

250 407

Graph 1. Glucometer Readings vs. Glucose Concentrations

Caption: The glucose concentrations read by the glucometer were plotted against the known

concentrations of glucose to find a line of best fit. The line of best fit shows the relationship

between the known glucose concentrations and the reading of the glucometer.

1 2 3 4 5 6 7 8 9 10 110

20

40

60

80

100

120

140

f(x) = 13.5 x − 25

Glucometer Readings vs. Known Glucose Concentration

Known Glucose Concentration (mg/dL)

Gluc

omet

er R

eadi

ng (m

g/dL

)

Table 2. Glucose Concentration in Pea Extract and Beano Solutions Over Time

Caption: This table shows the concentrations of glucose over two minute intervals in vials

containing solutions of Beano with three different concentrations of pea extract: 100%, 50%, and

25%.

Time (Minutes) Glucose in 100% Pea Extract (mg/dL)

Glucose in 50% Pea Extract (mg/dL)

Glucose in 25% Pea Extract (mg/dL)

2 low low Low4 32 23 Low6 56 58 238 74 77 2410 116 99 5112 137 134 5914 - - 83

Graph 2. Glucose Concentration vs. Time

Caption: This graph shows the amount of glucose concentration in each of the pea extract

solutions over time. The line of best fit represents the initial rate of reaction in each solution.

0 2 4 6 8 10 12 14 160

20

40

60

80

100

120

140

160

f(x) = 7.75 x − 29.5

f(x) = 13.15 x − 27f(x) = 13.5 x − 25

Glucose Concentration vs. Time

Time (minutes)

Gluc

ose

Conc

entr

ation

(mg/

dL)

Table 3. Rate of Reaction in 25%, 50%, and 100% Pea Extract Solutions

Caption: This table shows the rates of reaction in vary pea extract solutions.

Pea Extract Concentration Rate of Reaction

25% 7.75

50% 13.15

100% 13.5

Table 4. Glucose Concentration in 50% Pea Extract Solution at 10ᵒC, 25ᵒC, and 40ᵒC Over Time

Caption: This table shows the concentration of glucose over time when the reaction between

Beano and pea extract is carried out in three different temperatures: 10ᵒC, 25ᵒC, and 40ᵒC.

Time (minutes) 10ᵒC 25ᵒC 40ᵒC

1 Low Low Low

4 Low 23 31

6 Low 58 91

8 Low 77 149

10 21 99 22

12 32 134 250

14 42

16 51

18 67

Graph 3. Glucose Concentration vs. Time

Caption: This graph shows the glucose concentrations of vials in three different temperatures,

10C, 25C, and 40C over time. The line of best fit represents the initial rate of reaction between

the Beano and the 50% pea extract solution.

0 2 4 6 8 10 12 14 16 18 200

50

100

150

200

250

300f(x) = 28.45 x − 79

f(x) = 13.15 x − 27

f(x) = 5.55 x − 35.1

Gluose Concentration vs. Time

Time (minutes)

Gluc

ose

Conc

entr

ation

(mg/

dL)ᵒC

Table 5. Temperature and Reaction Rate

Caption: This table shows the varying temperatures the 50% Beano and pea extract solution were

placed in and the rate of the reaction.

Temperature (ᵒC) Rate of Reaction

10ᵒC 5.55

25ᵒC 13.15

40ᵒC 28.45

Discussion:

Overall this lab supported the hypotheses on the factors on the rate of reaction and

enzyme activity. Regarding the calibration of the glucometer to the pea-extract and Beano

solution, the glucometer read measurements much higher than the known concentrations. For

instance, the known concentration of glucose was 200 mg/dL, but the glucometer read 323

mg/dL. The correlation between the glucometer reading the known concentrations of glucose

was positive and linear. This shows that the glucometer overshot the actual amount of glucose in

the water-based media because it is designed to facto in the complex molecules in blood media

into its glucose reading.

The hypotheses regarding enzyme activity were affirmed. The hypothesis that increasing

the substrate concentration would increase the enzyme activity, increasing the rate of reaction

was averred. At low levels of substrate concentration, 25% pea extract, the initial rate of

reaction was about twice as slow as the initial rate of reaction when there was a substrate

concentration of 50% pea extract. The initial rates of reaction were not exactly proportionally

faster or slower based on concentration. 100% pea extract’s reaction rate, 13.5, was not double

that of 50% pea extract’s reaction rate, 13.15. The time it takes for the reaction to go to

completion may not be proportional between the solutions. After completion, however, after all

of the substrate is reacted with the enzyme, there would be proportional amount of glucose in

each pea extract solution. This is because there are only proportional amounts of substrate in

solution. For instance, there is ¾ less substrate in the 25% pea extract solution than in the 100%

pea extract solution. Thus, after completion, there would be ¾ less glucose in the 25% pea

extract vial than in the 100% pea extract vial.

Varying the temperature by 15ᵒC played a major role on the initial rates of reaction

between the enzymes in Beano and the pea extract. The results confirmed the hypothesis that at

higher temperatures, enzyme activity increases, which increases the rate of reaction. When the

reaction was carried out in 10ᵒC conditions, the initial rate of reaction was roughly 2.4 times

slower than when the reaction was carried out at 25ᵒC. When the reaction was carried out at 40ᵒC,

the reaction occurred at a rate 2.2 times faster than at 25ᵒC. This shows enzymatic activity is

directly related to temperature. With increasing temperature, the Beano enzymes interacted with

the pea extract substrate to produce glucose faster. Raising the temperature 15ᵒC had

approximately the same quantitative effect on the rate of reaction as lowering the temperature

15ᵒC. This further confirms that enzyme activity and reaction rates are very closely and directly

related to temperature.

Unfortunately, throughout this lab there was some room for error. There may have been

some inherent instrumental error in the glucometer that could have produced inaccurate readings

beyond the inaccuracy of media discrepancy. Also, the timing of glucose measurements could

have led to some error in results. The time that the glucose was measured was not always two

minutes on the dot. Sometimes, a few extra seconds passed and those seconds could have led to

different results. Another major room for error was in the water bath solutions. Maintaining an

environment of 40ᵒC is not easy. The heat could have escaped enough to lower the bathwater

enough to impact the rate of reaction. Conversely, the 10ᵒC bathwater was not exactly 10ᵒC the

whole time. The warmth of the room increased the bathwater temperature which would have

caused the reaction to occur faster than at exactly 10ᵒC. In short, there was a noticeable amount

of room for error in this experiment. However, the results still confirmed the hypotheses.

Conclusion:

Overall, this experiment fulfilled the predictions about factors affecting enzymatic

activity through the reaction of Beano and pea extract in glucose appearance. Beano is a

common medicine taken by people who experience uncomfortable pressure in their lower

stomach from bacteria breaking down the oligosaccharides in their large intestine because their

small intestine lacks the enzyme alpha-galactosidase that breaks down the oligosaccharides.

Beano contains the enzyme alpha-galactosidase. When Beano is consumed, the alpha-

galactosidase in Beano are released into the small intestine and the oligosaccharides can be

broken down in the small intestine. In this experiment, factors on the enzyme alpha-

galactosidase were measured by mixing Beano with pea-extract. It was found that increasing the

substrate concentration, the pea extract, increased the initial rate of reaction because there was

more substrate for the enzyme to interact with. It was also found that increasing temperature of

the Beano and pea-extract solution increased enzymatic activity because the substrate molecules

and enzyme molecules moved around faster, increasing their rate of collision, thus increasing the

rate of reaction. In summary, this experiment showed that the enzyme activity of alpha-

galactosidase in Beano follows that Collision Theory and the Kinetic Molecular Theory in that

enzyme activity increased with increasing temperature and that enzyme activity increased with

increasing concentration.

Citation:

1. "Collision Theory." Salve Regina University Department of Chemistry. Salve Regina

University. Web. 28 Feb 2013.

<http://www.salve.edu/academics/departments/chm/faculty.asp&xgt;.

2. Cooper, Geoffery. The Cell: A Molecular Approach. 2nd. Sunderland: Sinauer

Associates, 2000. Web.

3. Enzymes." The Royal Society of Chemists. The Royal Society of Chemists. Web. 20 Feb

2013. <http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm>.

4. . "Factors Effecting Enzyme Activity." Ohio State University Department of

Biochemistry. Ohio State University. Web. 28 Feb 2013. <http://class.fst.ohio-

state.edu/fst605/605p/EnzymeFactors.pdf>.

5. Bender, David. "The Effect of Substrate Concentration on Enzyme Activity." University

College London Dept of Chemistry. University College London. Web. 28 Feb 2013.

<http://www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm>.

6. Keiser, Joseph. PSU ChemTREK. Hayden McNeil. (Spring 2013): 6-1 – 6-15

7. 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 Nov; 39(5): 441-

5

8. Weinheimer, Alaina. Chem 113B Notebook.

9. Thomas, Rachel. Chem 113B Notebook.

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